Evaluation of topical ophthalmic ganciclovir gel for the treatment of dogs with experimentally induced ocular canine herpesvirus-1 infection

Eric C. Ledbetter Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Amanda M. Nicklin Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Chloe B. Spertus Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Matthew R. Pennington Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Gerlinde R. Van de Walle Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Hussni O. Mohammed Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Abstract

OBJECTIVE To determine the in vitro half maximal effective concentration (EC50) of ganciclovir for canine herpesvirus-1 (CHV-1) and to evaluate the efficacy of ganciclovir ophthalmic gel in dogs with experimentally induced ocular CHV-1 infection.

ANIMALS 10 specific pathogen–free adult Beagles.

PROCEDURES Cytotoxicity and EC50 of ganciclovir for CHV-1 were determined during in vitro experiments. During an in vivo experiment, dogs with experimentally induced ocular CHV-1 infections received 1 drop of 0.15% ganciclovir (ganciclovir group; n = 5) or artificial tear (control group; 5) ophthalmic gel in both eyes 5 times daily for 7 days, then 3 times daily for 7 days. For each dog, ophthalmic and confocal microscopic examinations were performed at predetermined times to determine severity of ocular disease and inflammation. Conjunctival swab specimens were collected at predetermined times for PCR assay analysis to determine CHV-1 shedding.

RESULTS No in vitro cytotoxic effects were observed for ganciclovir concentrations ≤ 500μM. The EC50 of ganciclovir for CHV-1 was 37.7μM. No adverse effects associated with ganciclovir were observed during the in vivo experiment. Mean ocular disease and inflammation scores for the ganciclovir group were significantly lower than those for the control group. Mean duration of CHV-1 shedding for the ganciclovir group (0.4 days) was significantly shorter than that for the control group (6.2 days).

CONCLUSIONS AND CLINICAL RELEVANCE Topical administration of 0.15% ganciclovir ophthalmic gel was well tolerated and effective in decreasing clinical disease scores, ocular tissue inflammation, and duration of viral shedding in dogs with experimentally induced ocular CHV-1 infection.

Abstract

OBJECTIVE To determine the in vitro half maximal effective concentration (EC50) of ganciclovir for canine herpesvirus-1 (CHV-1) and to evaluate the efficacy of ganciclovir ophthalmic gel in dogs with experimentally induced ocular CHV-1 infection.

ANIMALS 10 specific pathogen–free adult Beagles.

PROCEDURES Cytotoxicity and EC50 of ganciclovir for CHV-1 were determined during in vitro experiments. During an in vivo experiment, dogs with experimentally induced ocular CHV-1 infections received 1 drop of 0.15% ganciclovir (ganciclovir group; n = 5) or artificial tear (control group; 5) ophthalmic gel in both eyes 5 times daily for 7 days, then 3 times daily for 7 days. For each dog, ophthalmic and confocal microscopic examinations were performed at predetermined times to determine severity of ocular disease and inflammation. Conjunctival swab specimens were collected at predetermined times for PCR assay analysis to determine CHV-1 shedding.

RESULTS No in vitro cytotoxic effects were observed for ganciclovir concentrations ≤ 500μM. The EC50 of ganciclovir for CHV-1 was 37.7μM. No adverse effects associated with ganciclovir were observed during the in vivo experiment. Mean ocular disease and inflammation scores for the ganciclovir group were significantly lower than those for the control group. Mean duration of CHV-1 shedding for the ganciclovir group (0.4 days) was significantly shorter than that for the control group (6.2 days).

CONCLUSIONS AND CLINICAL RELEVANCE Topical administration of 0.15% ganciclovir ophthalmic gel was well tolerated and effective in decreasing clinical disease scores, ocular tissue inflammation, and duration of viral shedding in dogs with experimentally induced ocular CHV-1 infection.

Canine herpesvirus-1 is a varicellovirus of the subfamily Alphaherpesvirinae with a worldwide distribution and host range restricted to canids.1–3 It was originally identified as an etiology of fetal and neonatal systemic infections. Now, CHV-1 is also recognized as a pathogen of adult dogs that is associated with a variety of dermatologic, genital, respiratory, and ocular conditions.4–7 In domestic adult dogs, ocular lesions associated with primary and recurrent CHV-1 infection include blepharitis, conjunctivitis, ulcerative keratitis, and nonulcerative keratitis.8,9

Ganciclovir is a synthetic nucleoside analog of guanosine that is selectively phosphorylated by virus-encoded enzymes (eg, thymidine kinase or protein kinase) into ganciclovir monophosphate.10,11 The phosphorylated form of ganciclovir is further phosphorylated by both viral and cellular thymidine kinases of virus-infected cells to the active metabolite ganciclovir triphosphate.12 Ganciclovir triphosphate accumulates only in virus-infected cells; thus, it is fairly nontoxic to healthy host cells. Ganciclovir has activity against a broad spectrum of human herpesviruses and adenoviruses.13,14 Ganciclovir can be administered by the I V, intravitreal, and PO routes and is used for the treatment of several types of human cytomegalovirus infections, including retinitis, pneumonitis, colitis, and encephalitis.15

Ganciclovir is water soluble, which facilitates its formulation as an aqueous preparation for topical ocular application.16 A 0.15% ganciclovir aqueous gel is commercially available and marketed in the United States and Europe.17 The solubilization of ganciclovir as an aqueous gel extends drug contact time on the ocular surface, enhances tissue absorption, improves tolerability, and results in extended therapeutic concentrations in ocular tissues and fluids.18

In human medicine, topical ganciclovir gel is a clinically effective antiviral agent used for the treatment of various ocular herpesvirus infections, such as HSV-1 keratitis, herpes zoster ophthalmicus, cytomegalovirus corneal endotheliitis and anterior uveitis, and human herpesvirus-6 corneal endotheliitis.19–23 Additionally, evidence suggests that ganciclovir gel is an effective treatment for human adenovirus conjunctivitis.24,25 In veterinary medicine, ganciclovir ophthalmic gel has been successfully used to treat a dog with CHV-1 dendritic ulcerative keratitis.a However, the in vitro antiviral efficacy of ganciclovir against ocular CHV-1 isolates and the in vivo effects of treatment with ganciclovir ophthalmic gel in dogs with experimentally induced ocular CHV-1 infection have not been described. Therefore, the purpose of the study reported here was to determine the in vitro antiviral activity of ganciclovir against CHV-1 and to evaluate the treatment effects of ganciclovir ophthalmic gel in dogs with experimentally induced ocular CHV-1 infection.

Materials and Methods

Study design and animals

The study consisted of in vitro and in vivo experiments. During in vitro experiments, the cytotoxic and antiviral effects of ganciclovir at various concentrations were determined for MDCK cell cultures that were and were not infected with CHV-1, respectively. During the in vivo experiment, latent CHV-1 infection was experimentally induced in 10 adult dogs, which were then randomly assigned to be treated with topical ganciclovir or artificial tears (control). All in vivo procedures were approved by the Cornell University Animal Care and Use Committee and were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.26

Ten 2.5-year-old specific pathogen–free laboratory Beagles were used for the in vivo experiment. All dogs were seronegative for antibodies against CHV-1. Dogs were housed in an isolation facility for the duration of the study. They were maintained individually in runs that prevented direct contact between dogs. The dogs were acclimated to the housing facilities for a minimum of 12 weeks before initiation of study procedures.

In vitro evaluation of the cytotoxic and anti–CHV-1 effects of ganciclovir

Madin-Darby canine kidney cells were used for all in vitro procedures. The cells were maintained in cell line medium, which consisted of DMEMb with 1 g/L glucose, l-glutamine, and sodium pyruvate that contained 10% fetal bovine serum,c and penicillin (200 U/mL)-streptomycind (200 μg/mL) at 37°C and 5% CO2.

Cell viability was assessed by use of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.e Briefly, MDCK cells were treated with ganciclovirf dissolved in 0.1M hydrochloride at serial 2-fold dilutions that ranged from 0 to 2,000μM and incubated for 68 hours. There were at least 3 replicates for each dilution. Then, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dissolved in DMEM was added to the ganciclovir-treated cells, and the cell cultures were incubated for an additional 4 hours to allow for formazan crystal formation. The formazan crystals were dissolved with an equal volume of solubilization solutione (10% Triton X-100 and 0.1 N HCl in isopropanol), and light absorbance was spectrophotometrically measured at 570 nm. For each ganciclovir-treated cell culture, the relative cell viability was calculated as follows: (optical density of drug dilution at 570 nm – optical density of drug dilution at 690 nm)/(optical density of nontreated at 570 nm – optical density of drug dilution at 690 nm) × 100%.

The EC50 of ganciclovir for CHV-1 was determined as described.27 Briefly, 10 4 MDCK cells were added to each well of a 96-well plate and cultured overnight (approx 20 hours). Then, 100 plaque-forming units of a previously described field strain of CHV-128 were added to each well. The cells within each well were treated with ganciclovir at serial 2-fold dilutions that ranged from 0 to 800μM. There were at least 3 replicates for each dilution. Cells were cultured approximately 72 hours or until cytopathic effect was visible in untreated control cells. Cells were fixed with 90% ethanol and stained with crystal violet. The percentage of wells that contained cytopathic effect was determined for each ganciclovir dilution.

In vivo experiment

Experimental induction of ocular CHV-1 infection— Twelve months prior to study initiation, a latent ocular CHV-1 infection was experimentally induced in each dog by use of the topical ocular drop method as described.28 Briefly, both eyes of each dog were topically inoculated with 2 × 105 TCID50 of a CHV-1 field strain (CHV-1-Duk) isolated from corneal specimens of a dog with dendritic ulcerative keratitis that was treated at the Cornell University College of Veterinary Medicine Hospital for Animals. The eyelid of each eye was held closed immediately after virus instillation and gently massaged for 60 seconds. Each dog was monitored for 30 days after CHV-1 inoculation to confirm the development of primary ocular CHV-1 infection, during which time clinical ophthalmic examinations were performed at 3-day intervals and serum neutralizing anti–CHV-1 antibody titers were determined at 15-day intervals.

Treatment administration—Each dog was randomly assigned by means of a random number generator to receive either ganciclovir (ganciclovir group; n = 5) or artificial tear (control group; 5) ophthalmic gel, and the study had a duration of 30 days (days 1 through 30). For each dog, the latent ocular CHV-1 infection was experimentally recrudesced as described.29 Briefly, beginning on day 1, each dog received prednisolone (3.0 mg/kg, PO, q 24 h) for 7 consecutive days. Beginning on day 4, dogs in the ganciclovir group received 1 drop of 0.15% ganciclovir ophthalmic gel,g whereas dogs in the control group received 1 drop of artificial tear ophthalmic gelh in each eye 5 times daily (at 3-hour intervals between 8:00 am and 8:00 pm) for 7 days (days 4 through 10) and then 3 times daily (at 6-hour intervals between 8:00 am and 8:00 pm) for another 7 days (days 11 through 17). The labels on the assigned treatment preparations were concealed such that the persons (AMN and CBS) who administered the treatments and all investigators remained unaware of (were blinded to) the treatment group assignment for each dog.

Ophthalmic examination—A physical examination and complete ophthalmic examination, which included slit-lamp biomicroscopy,i indirect ophthalmoscopy, a Schirmer I tear test,j and corneal application of lissamine green stain,k were performed on each dog before study initiation. Slit-lamp biomicroscopy was performed on both eyes before and after application of lissamine green stain at 2-day intervals throughout the duration of the study. Ophthalmic examination findings were quantified by use of a previously described modified ocular surface disease clinical scoring system.28 Briefly, for each eye, the severity of blepharospasm, ocular discharge, conjunctival hyperemia, chemosis, conjunctival ulceration, and corneal epithelial ulceration was assessed on a 4-point scale. For all variables except conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = mild, 2 = moderate, and 3 = severe. For conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = punctate ulcerations, 2 = ≥ 1 linear or dendritic ulceration, and 3 = geographic ulcerations. On each examination day, a single cumulative ocular surface disease clinical score (ocular clinical disease score) was calculated for each dog. For dogs that had nonsymmetric ocular disease on any given examination, the highest score was used for statistical analyses.

In vivo confocal microscopic examination—For each dog, in vivo confocal microscopic examinationsl of the cornea and conjunctiva of both eyes were performed by use of a 63X objectivem and 400-μm field lens immediately before study initiation and on day 10. For each eye, the examination was performed following the application of 1 drop of topical anesthetic (0.5% proparacaine ophthalmic solution) and several drops of contact gel to the ocular surface. A sterile, single-use polymethylmethacrylate capn mounted on the microscope lens was positioned perpendicular to and in slight contact with the ocular surface. The polymethylmethacrylate cap was changed after each dog was examined.

Multipoint corneal and conjunctival imaging was performed with a combination of manual and automated image acquisition modes. Following acquisition, digitized images were analyzed for pathological lesions. Images of standardized anatomic locations were acquired, and an investigator (ECL) who was blinded to the treatment group assignment of each dog assigned a leukocyte infiltration score to each image as described.27 Images of the basal corneal epithelium acquired 1.0 mm anterior to the 12 o'clock superior limbal position were used to assess keratitis. Images of the surface conjunctival epithelium acquired 1.0 mm posterior to the 12 o'clock superior limbal position were used to assess conjunctivitis. Leukocytes were quantified (number of leukocytes/mm2 of tissue) in 3 corneal images from each eye by use of semiautomated cell-counting software.o Leukocytes were also quantified in 3 conjunctival images from each eye; however, because distinct leukocyte cell borders are difficult to distinguish in conjunctival images, a 4-point semiquantitative scoring system was used to describe conjunctival infiltrates, where 0 = absent, 1 = mild, 2 = moderate, and 3 = marked.

Sample collection—From each dog, blood samples for a CBC (approx 2.0 mL) and serum biochemical profile (approx 2.0 mL) were collected by peripheral venipuncture immediately before study initiation and on days 15 and 30. Following clinical ocular disease scoring, but before lissamine green stain application, conjunctival swab specimens for detection of CHV-1 by PCR assay were collected from both eyes by brushing sterile polyester-tipped swabs across the conjunctival fornices. The swab specimens were collected at 3-day intervals for the duration of the study and stored in sterile tubes at −80°C until analysis.

CHV-1 real-time PCR assay—A real-time PCR assay was used to detect CHV-1 (viral shedding) in conjunctival swab specimens. The CHV-1–specific primers and probes used and the assay conditions have been described elsewhere.29 For each swab specimen, DNA was extracted with a commercial DNA extraction kitp and use of a 96-well magnetic bead–based process.q

Statistical analysis

For the in vitro experiment, indices of cytotoxicity and anti–CHV-1 efficacy were compared between ganciclovir-treated and control cells by use of Student t tests. For the in vivo experiment, regression analysis was used to evaluate the association between the ocular clinical disease score and treatment group (ganciclovir and control) over time and within each study day; the appropriate transformation of time was considered prior to the analysis. Student t tests were used to compare the mean confocal conjunctivitis and keratitis (leukocyte infiltration) scores between the 2 treatment groups. Logistic regression was used to assess the effects of treatment group and study day on CHV-1 shedding (yes or no). All statistical analyses were performed with a statistical software package,r and values of P ≤ 0.05 were considered significant.

Results

In vitro cytotoxic and anti–CHV-1 effects of ganciclovir

Ganciclovir had significant cytotoxic effects on MDCK cell cultures only at the 2 highest concentrations (1,000 and 2,000μM) evaluated. Mean ± SD percentage cell viability of ganciclovir-treated cells relative to that of control cells was 92 ± 5% and 75 ± 8% at drug concentrations of 1,000 and 2,000μM, respectively. Ganciclovir did not significantly affect MDCK cell viability at concentrations < 1,000μM (Figure 1). The mean ± SD EC50 of ganciclovir against CHV-1 was 37.7 ± 1.1μ M (R2 = 0.949; Figure 2).

Figure 1—
Figure 1—

Mean ± SD relative cell viability for MDCK cell cultures that were treated with ganciclovir at serial 2-fold dilutions ranging from 0 to 2,000μM. There were at least 3 replicates for each ganciclovir dilution and the untreated control. Madin-Darby canine kidney cell cultures maintained in cell line medium, which consisted of DMEM with 1 g/L of glucose, l-glutamine, and sodium pyruvate that contained 10% fetal bovine serum and penicillin (200 U/mL)-streptomycin (200 μg/mL), were incubated with the assigned ganciclovir dilution for 68 hours. Then 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dissolved in DMEM was added to each culture, and the cultures were incubated for an additional 4 hours to allow formazan crystal formation. The formazan crystals were dissolved with an equal volume of solubilization solution, and light absorbance was spectrophotometrically measured at 570 nm. Cell viability for untreated control cultures was set at 100% (dotted line). For each ganciclovir-treated cell culture, the relative cell viability was calculated as follows: (cell viability for that culture/100) × 100%. *Mean differs significantly (P ≤ 0.05) from 100%.

Citation: American Journal of Veterinary Research 79, 7; 10.2460/ajvr.79.7.762

Figure 2—
Figure 2—

Mean ± SD percentage of CHV-1–infected MDCK cell culture replicates that contained cytopathic effect following incubation with ganciclovir at serial 2-fold dilutions that ranged from 0 to 800μM. There were at least 3 replicates for each ganciclovir dilution. The CHV-1–infected cells were incubated with the assigned ganciclovir dilution for approximately 72 hours or until cytopathic effect was visible in control cells that were not treated with ganciclovir. The percentage of replicates that contained cytopathic effect was determined for each ganciclovir dilution. The solid line represents the line of best fit for the data and was used to determine the EC50 of ganciclovir for CHV-1 (dotted lines), which was 37.7 μM for this data set.

Citation: American Journal of Veterinary Research 79, 7; 10.2460/ajvr.79.7.762

In vivo efficacy of ganciclovir

Immediately before study initiation on day 1 (baseline), no abnormalities were detected on the basis of results of the physical examination, CBC, and serum biochemical profile for any dog. Likewise, no abnormalities were detected in either eye during the baseline ophthalmic examination for any dog. For both eyes of each dog, Schirmer I tear test results were greater than the reference cutoff (> 15 mm/min), corneal and conjunctival retention of lissamine green stain was not present, and no conjunctival or corneal abnormalities (leukocyte infiltration) were detected by in vivo confocal microscopy at baseline.

No overt systemic abnormalities were observed in any dog at any time during the study. No adverse effects associated with topical administration of either the ganciclovir or artificial tear ophthalmic gel were observed. Results of CBCs and serum biochemistry profiles were unremarkable for blood samples collected from all dogs on days 15 and 30.

For all dogs in both the ganciclovir and control groups, recurrent ocular CHV-1 infection was clinically evident by day 4 and was characterized by intermittent blepharospasm, conjunctival hyperemia, chemosis, and ocular discharge. The mean ocular clinical disease score was curvilinear over time for both treatment groups. It increased fairly rapidly and then declined slowly (Figure 3). The mean ocular clinical disease score peaked on day 6 for the ganciclovir group (mean ± SD score, 2.2 ± 1.2) and on day 8 for the control group (mean ± SD score, 4.2 ± 1.0). Beginning on day 8 and for the remainder of the study duration, the mean ocular clinical disease score for the ganciclovir group was significantly (P ≤ 0.001) lower than that for the control group. During that time, the mean ocular clinical disease score for the ganciclovir group was, on average, 1.1 less than that for the treatment group (ie, the control group was the referent and the regression coefficient for ganciclovir group was 1.146). The intercept for the regression analysis did not differ significantly from 0; therefore, it was deleted from the analysis.

Figure 3—
Figure 3—

Mean ± SD cumulative ocular clinical disease score over time for adult dogs with experimentally induced recurrent ocular CHV-1 infection that, beginning on day 4, received 1 drop of 0.15% ganciclovir ophthalmic ointment gel (ganciclovir group; n = 5; gray bars) or 1 drop of artificial tear ophthalmic ointment gel (control group; 5; black bars) in each eye 5 times daily (at approx 3-hour intervals) for 7 days (days 4 through 10) and then 3 times daily (at approx 6-hour intervals) for another 7 days (days 11 through 17). Beginning on day 1, dogs with latent CHV-1 infection were treated with prednisolone (3.0 mg/kg, PO, q 24 h) for 7 days to make the infection recrudesce. At 2-day intervals throughout the 30-day study period, the severity of blepharospasm, ocular discharge, conjunctival hyperemia, chemosis, conjunctival ulceration, and corneal epithelial ulceration was assessed on a 4-point scale. For all variables except conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = mild, 2 = moderate, and 3 = severe. For conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = punctate ulcerations, 2 = ≥ 1 linear or dendritic ulcerations, and 3 = geographic ulcerations. For each dog, a single cumulative ocular clinical disease score was calculated at each evaluation. For dogs that had nonsymmetric ocular disease on any given evaluation, the highest score was used for statistical analyses.

Citation: American Journal of Veterinary Research 79, 7; 10.2460/ajvr.79.7.762

For the control group, CHV-1 was detected in conjunctival swab specimens obtained from 1 dog on day 3, 3 dogs on day 6, 4 dogs on day 9, 3 dogs on day 12, and 2 dogs on day 15. For the ganciclovir group, CHV-1 was detected in the swab specimens obtained from 2 dogs on day 6. The virus was not detected in swab specimens obtained from any dog on the other study days. The mean ± SD number of days that CHV-1 was detected was 0.4 ± 0.5 days for the ganciclovir group and 2.6 ± 1.7 days for the control group. The mean ± SD duration of CHV-1 shedding was 0.4 ± 0.5 days for the ganciclovir group and 6.2 ± 4.7 days for the control group. The odds that dogs in the ganciclovir group would shed CHV-1 (OR, 0.1; 95% confidence interval, 0.02 to 0.5; P = 0.005) was only 10% that for dogs in the control group.

In vivo confocal microscopy revealed leukocyte infiltration of the bulbar conjunctival epithelium and corneal basal epithelium in all 10 dogs on day 10 (Figure 4). On that day, the mean ± SD conjunctivitis score for the ganciclovir group (1.0 ± 0.9) was significantly (P < 0.001) less than that for the control group (2.8 ± 0.4). Likewise, the mean ± SD keratitis score for the ganciclovir group (103 ± 70 leukocytes/mm2) was significantly (P < 0.001) less than that for the control group (344 ± 130 leukocytes/mm2).

Figure 4—
Figure 4—

Representative in vivo confocal photomicrographs of the bulbar conjunctival epithelium (A and B) and corneal basal epithelium (C and D) obtained on day 10 for various dogs in the ganciclovir (B and D) and control (A and C) groups described in Figure 3. Leukocytes infiltrating the tissue appear as highly reflective and irregularly shaped cells. Leukocyte infiltration was considered moderate for dogs in the control group and mild for dogs in the ganciclovir group. Bar = 50 μm. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 79, 7; 10.2460/ajvr.79.7.762

Discussion

Results of the present study indicated that ganciclovir had in vitro activity against CHV-1 and effectively decreased the severity of clinical ocular disease and inflammation of ocular tissues when topically administered to dogs with experimentally induced recurrent ocular CHV-1 infection. The 0.15% ganciclovir ophthalmic gel evaluated was well tolerated by the study dogs, as evidenced by the fact that none of the dogs developed clinical evidence of local or systemic toxicosis. Those findings suggested that 0.15% ganciclovir ophthalmic gel was a safe and effective treatment option for dogs with ocular CHV-1 infections. A benefit of the ganciclovir ophthalmic gel evaluated in this study is that it is commercially marketed and readily available as a topical ophthalmic product and does not require pharmaceutical compounding as do many other ophthalmic antivirals used for topical application in veterinary medicine.30

Other antivirals used for topical treatment of dogs with ocular CHV-1 infections include idoxuridine, trifluridine, and cidofovir ophthalmic solutions.31,32 Clinically, 0.15% ganciclovir ophthalmic gel has been successfully used to treat a dog with CHV-1 dendritic ulcerative keratitis.a The dog of that reporta developed keratitis, which, on the basis of results of a PCR assay, was determined to be caused by CHV-1 while the dog was being treated with topical formulations of cyclosporine and tacrolimus for keratoconjunctivitis sicca. The ganciclovir gel was topically applied to both eyes 5 times daily for 4 weeks.a During a recheck ophthalmic examination performed 1 month later, the corneal ulcers had resolved, and PCR assay results for a conjunctival swab specimen were negative for CHV-1.a

In other studies, dogs with experimentally induced ocular CHV-1 infection were topically treated with 0.5% cidofovir27 and 1.0% trifluridine33 ophthalmic solutions. Twice-daily administration of cidofovir for 14 days reduced the duration of CHV-1 shedding from ocular tissues but exacerbated clinical ocular disease and corneoconjunctival inflammation.27 The signs of marked local ocular toxicosis associated with the concentration and formulation of cidofovir ophthalmic solution evaluated in that study27 preclude its routine clinical use in dogs with ocular CHV-1 infections. In the other study,33 dogs received trifluridine 6 times daily for 2 days and then 4 times daily for the subsequent 12 days. The trifluridine-treated dogs of that study33 tolerated the drug well and had decreased ocular clinical disease severity and decreased incidence and duration of ocular CHV-1 shedding, compared with control dogs that were treated with a placebo solution.

On the basis of the veterinary literature33 and results of the present study, both trifluridine 1.0% ophthalmic solution and ganciclovir 0.15% ophthalmic gel appear to be acceptable treatment options for dogs with clinical ocular CHV-1 infections. The administration frequency used for ganciclovir in the present study (1 drop 5 times daily for 7 days and then 3 times daily for an additional 7 days) was selected to approximate the general treatment recommendation for human patients with acute herpetic keratitis, which is 1 drop 5 times daily until corneal ulcers are healed and then 1 drop 3 times daily for another week.11 In vitro synergism between trifluridine and ganciclovir against HSV-1 has been described, and when the 2 drugs are used in combination, the anti-HSV-1 effect is achieved at lower doses of both drugs than when either drug is used alone.34 It is currently unknown whether a similar phenomenon occurs against CHV-1, but if it does, combination treatment might allow for a decrease in the frequency of drug application. Thus, topical administration of various combinations of antiviral agents warrants investigation in dogs with ocular CHV-1 infections.

The efficacy of topical ganciclovir administration for the treatment of HSV-1 keratitis is well established in both preclinical and clinical studies.35–40 In rabbits with experimentally induced HSV-1 keratitis, ganciclovir ameliorated clinical ocular disease and reduced the duration of viral shedding in the tear film without causing any evidence of ocular toxicosis.35–37 In one of those studies,35 no significant differences in treatment efficacy were observed between rabbits that were treated with a 0.1% ganciclovir formulation and those treated with a 1.0% ganciclovir formulation. In another study18 in which rabbits with experimentally induced HSV-1 keratitis were treated with 0.2%, 0.05%, or 0.0125% ganciclovir gel, corneal ulcer healing was quickest for eyes treated with the 0.05% gel. The safety, tolerability, and efficacy of 0.15% ganciclovir ophthalmic gel for the treatment of human patients with acute HSV-1 keratitis have also been evaluated,38–40 and a recent review41 of the human medical literature suggests that ganciclovir ophthalmic gel should be considered one of the first-line treatments for patients with acute ocular herpetic infections.

The EC50 of ganciclovir for the CHV-1 strain used in this study was 37.7μM, which is greater than the EC50 of ganciclovir for other alphaherpesviruses such as certain strains of HSV-134 (EC50 range, 0.4 to 1.6μM), feline herpesvirus type-142 (EC50, 5.2μM), and equine herpesvirus type-143 (EC50 range, 0.39 to 1.56μM). The fairly high EC50 calculated in this study was consistent with the EC50 of ganciclovir for a CHV-1 strain isolated from the respiratory tract of a dog (> 32 μg/mL) and greater than the EC50 of ganciclovir for several other equine, human, swine, bovine, and feline herpesvirus isolates that were concurrently evaluated in another study.44 However, owing to the low in vitro toxicity of ganciclovir in MDCK cells and the lack of adverse effects observed in vivo for the ganciclovir-treated dogs of the present study, the fairly high EC50 of ganciclovir against CHV-1 would not be expected to preclude its clinical use in dogs with ocular CHV-1 infections.

In the present study, topical administration of 0.15% ganciclovir ophthalmic gel was well tolerated and effective in decreasing clinical disease scores, ocular tissue inflammation, and duration of viral shedding in dogs with experimentally induced ocular CHV-1 infection. The concurrent administration of prednisolone to dogs during a portion of the in vivo experiment may have affected some of the observed variables, such as the absolute ocular clinical disease scores and confocal microscopy leukocyte infiltration scores. However, dogs in both the ganciclovir and control groups received the same dosage of prednisolone to recrudesce the experimentally induced latent CHV-1 infection; thus, comparisons between the 2 treatment groups were valid. Additionally, the established experimental model of ocular CHV-1 infection parallels naturally acquired disease, which commonly develops in dogs during treatment with immunosuppressive agents.2 7,33 Thus, topical administration of ganciclovir ophthalmic gel appears to be a safe and effective treatment option for dogs with ocular CHV-1 infections.

Acknowledgments

Supported by the Cornell University Research Grants Program in Animal Health. The funding agency did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

ABBREVIATIONS

CHV-1

Canine herpesvirus-1

DMEM

Dulbecco modified Eagle medium

EC50

Half maximal effective concentration

HSV-1

Herpes simplex virus-1

MDCK

Madin-Darby canine kidney

Footnotes

a.

Appelboam H. A case of bilateral herpetic keratitis in a dog (abstr). Vet Ophthalmol 2014;17:E27–E28.

b.

Cell Grow, Mediatech Inc, Manassas, Va.

c.

Atlanta Biologicals, Flowery Branch, Ga.

d.

Life Technologies, Grand Island, NY.

e.

Sigma-Aldrich, St Louis, Mo.

f.

Ganciclovir, Sigma-Aldrich, St Louis, Mo.

g.

Zirgan, Bausch & Lomb Inc, Tampa, Fla.

h.

Genteal lubricant eye gel, Novartis Pharmaceutical Corp, East Hanover, NJ.

i.

Kowa SL-17, Kowa Co, Tokyo, Japan.

j.

Schirmer tear test standardized sterile strips, Intervet Inc, Summit, NJ.

k.

Lissamine green ophthalmic strips, Contacare Ophthalmics & Diagnostics, Gujarat, India.

l.

Heidelberg Engineering, Dossenheim, Germany.

m.

Carl Zeiss Meditec AG, Jena, Germany.

n.

TomoCap, Heidelberg Engineering, Dossenheim, Germany.

o.

Rostock Cornea Module Software, version 1.3.3, Heidelberg Engineering, Dossenheim, Germany.

p.

AM 1840, Life Technologies, Grand Island, NY.

q.

Mag Max 96, Life Technologies, Grand Island, NY.

r.

SPSS, version 24, IBM Statistical Software, White Plain, NY.

References

  • 1. Rémond M, Sheldrick P, Lebreton F, et al. Gene organization in the UL region and inverted repeats of the canine herpesvirus genome. J Gen Virol 1996;77:3748.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Krogenæs A, Rootwelt V, Larsen S, et al. A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population. Theriogenology 2012;78:153158.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Babaei H, Akhtardanesh B, Ghanbarpour R, et al. Serological evidence of canine herpesvirus-1 in dogs of Kerman city, south-east of Iran. Transbound Emerg Dis 2010;57:348351.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Carmichael LE, Squire RA, Krook L. Clinical and pathologic features of a fatal viral disease of newborn pups. Am J Vet Res 1965;26:803814.

    • Search Google Scholar
    • Export Citation
  • 5. De Palma VE, Ayala MA, Gobello C, et al. An atypical clinical presentation for the first isolation of canid herpesvirus 1 in Argentina. Arq Bras Med Vet Zootec 2010;62:12671270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Hill H, Maré CJ. Genital disease in dogs caused by canine herpesvirus. Am J Vet Res 1974;35:669672.

  • 7. Karpas A, Garcia FG, Calvo F, et al. Experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally infected dogs. Am J Vet Res 1968;29:12511257.

    • Search Google Scholar
    • Export Citation
  • 8. Ledbetter EC, Kim SG, Dubovi EJ. Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony. Vet Ophthalmol 2009;12:242247.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Ledbetter EC. Canine herpesvirus-1 ocular diseases of mature dogs. N Z Vet J 2013;61:193201.

  • 10. Crumpacker CS. Ganciclovir. N Engl J Med 1996;335:721729.

  • 11. Sahin A, Hamrah P. Acute herpetic keratitis: what is the role for ganciclovir ophthalmic gel? Ophthalmol Eye Dis 2012;4:2334.

  • 12. Matthews T, Boehme R. Antiviral activity and mechanism of action of ganciclovir. Rev Infect Dis 1988;10(suppl 3):S490S494.

  • 13. Field AK, Davies ME, DeWitt C, et al. 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci U S A 1983;80:41394143.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Huang J, Kadonosono K, Uchio E. Antiadenoviral effects of ganciclovir in types inducing keratoconjunctivitis by quantitative polymerase chain reaction methods. Clin Ophthalmol 2014;8:315320.

    • Search Google Scholar
    • Export Citation
  • 15. Kotton CN. CMV: prevention, diagnosis and therapy. Am J Transplant 2013;13(suppl 3):2440.

  • 16. Croxtall JD. Ganciclovir ophthalmic gel 0.15%: in acute herpetic keratitis (dendritic ulcers). Drugs 2011;71:603610.

  • 17. Tabbara KF, Al Balushi N. Topical ganciclovir in the treatment of acute herpetic keratitis. Clin Ophthalmol 2010;4:905912.

  • 18. Castela N, Vermerie N, Chast F, et al. Ganciclovir ophthalmic gel in herpes simplex virus rabbit keratitis: intraocular penetration and efficacy. J Ocul Pharmacol 1994;10:439451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Kaufman HE, Haw WH. Ganciclovir ophthalmic gel 0.15%: safety and efficacy of a new treatment for herpes simplex keratitis. Curr Eye Res 2012;37:654660.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Aggarwal S, Cavalcanti BM, Pavan-Langston D. Treatment of pseudodendrites in herpes zoster ophthalmicus with topical ganciclovir 0.15% gel. Cornea 2014;33:109113.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Koizumi N, Miyazaki D, Inoue T, et al. The effect of topical application of 0.15% ganciclovir gel on cytomegalovirus corneal endotheliitis. Br J Ophthalmol 2017;101:114119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Wong JX, Agrawal R, Wong EP, et al. Efficacy and safety of topical ganciclovir in the management of cytomegalovirus (CMV)-related anterior uveitis. J Ophthalmic Inflamm Infect 2016; 6:10.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Yokogawa H, Kobayashi A, Yamazaki N, et al. Identification of cytomegalovirus and human herpesvirus-6 DNA in a patient with corneal endotheliitis. Jpn J Ophthalmol 2013;57:185190.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Yabiku ST, Yabiku MM, Bottós KM, et al. Ganciclovir 0.15% ophthalmic gel in the treatment of adenovirus keratoconjunctivitis. Arq Bras Oftalmol 2011;74:417421.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Ozen S, Ozer MA. Ganciclovir ophthalmic gel treatment shortens the recovery time and prevents complications in the adenoviral eye infection. Int Ophthalmol 2017;37:245249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Association for Research in Vision and Ophthalmology. Statement for the use of animals in ophthalmic and vision research. Available at: www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-visual-research/. Accessed Jan 9, 2018.

    • Search Google Scholar
    • Export Citation
  • 27. Ledbetter EC, Spertus CB, Pennington MR, et al. In vitro and in vivo evaluation of cidofovir as a topical ophthalmic antiviral for ocular canine herpesvirus-1 infections in dogs. J Ocul Pharmacol Ther 2015;31:642649.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Ledbetter EC, Dubovi EJ, Kim SG, et al. Experimental primary ocular canine herpesvirus-1 infection in adult dogs. Am J Vet Res 2009;70:513521.

  • 29. Ledbetter EC, da Silva EC, Kim SG, et al. Frequency of spontaneous canine herpesvirus-1 reactivation and ocular viral shedding in latently infected dogs and canine herpesvirus-1 reactivation and ocular viral shedding induced by topical administration of cyclosporine and systemic administration of corticosteroids. Am J Vet Res 2012;73:10791084.

    • Search Google Scholar
    • Export Citation
  • 30. Thomasy SM, Maggs DJ. A review of antiviral drugs and other compounds with activity against feline herpesvirus type 1. Vet Ophthalmol 2016;19 (suppl 1):119130.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Ledbetter EC, Riis RC, Kern TJ, et al. Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs. J Am Vet Med Assoc 2006;229:376384.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Gervais KJ, Pirie CG, Ledbetter EC, et al. Acute primary canine herpesvirus-1 dendritic ulcerative keratitis in an adult dog. Vet Ophthalmol 2012;15:133138.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Spertus CB, Mohammed HO, Ledbetter EC. Effects of topical ocular application of 1% trifluridine ophthalmic solution in dogs with experimentally induced recurrent ocular canine herpesvirus-1 infection. Am J Vet Res 2016;77:11401147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Hobden JA, Kumar M, Kaufman HE, et al. In vitro synergism of trifluorothymidine and ganciclovir against HSV-1. Invest Ophthalmol Vis Sci 2011;52:830833.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Trousdale MD, Nesburn AB, Willey DE, et al. Efficacy of BW759 (9-[[2-hydroxy-1(hydroxymethyl)ethoxy]methyl] guanine) against herpes simplex virus type 1 keratitis in rabbits. Curr Eye Res 1984;3:10071015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Shiota H, Naito T, Mimura Y. Anti-herpes simplex virus (HSV) effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea. Curr Eye Res 1987;6:241245.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Smith KO, Hodges SL, Kennell WL, et al. Experimental ocular herpetic infections in rabbits. Treatment with 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine. Arch Ophthalmol 1984;102:778781.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Colin J, Hoh HB, Easty DL, et al. Ganciclovir ophthalmic gel (Virgan; 0.15%) in the treatment of herpes simplex keratitis. Cornea 1997;16:393399.

    • Search Google Scholar
    • Export Citation
  • 39. Chou TY, Hong BY. Ganciclovir ophthalmic gel 0.15% for the treatment of acute herpetic keratitis: background, effectiveness, tolerability, safety, and future applications. Ther Clin Risk Manag 2014;10:665681.

    • Search Google Scholar
    • Export Citation
  • 40. Wilhelmus KR. Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev 2015;1:CD002898.

    • Search Google Scholar
    • Export Citation
  • 41. Tsatsos M, MacGregor C, Athanasiadis I, et al. Herpes simplex virus keratitis: an update of the pathogenesis and current treatment with oral and topical antiviral agents. Clin Experiment Ophthalmol 2016;44:824837.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Maggs DJ, Clarke HE. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. Am J Vet Res 2004;65:399403.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Garré B, van der Meulen K, Nugent J, et al. In vitro susceptibility of six isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet. Vet Microbiol 2007;122:4351.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Smith KO, Galloway KS, Hodges SL, et al. Sensitivity of equine herpesviruses 1 and 3 in vitro to a new nucleoside analogue, 9-[[2-hydroxy-1-(hydroxymethyl) ethoxy] methyl] guanine. Am J Vet Res 1983;44:10321035.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Mean ± SD relative cell viability for MDCK cell cultures that were treated with ganciclovir at serial 2-fold dilutions ranging from 0 to 2,000μM. There were at least 3 replicates for each ganciclovir dilution and the untreated control. Madin-Darby canine kidney cell cultures maintained in cell line medium, which consisted of DMEM with 1 g/L of glucose, l-glutamine, and sodium pyruvate that contained 10% fetal bovine serum and penicillin (200 U/mL)-streptomycin (200 μg/mL), were incubated with the assigned ganciclovir dilution for 68 hours. Then 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dissolved in DMEM was added to each culture, and the cultures were incubated for an additional 4 hours to allow formazan crystal formation. The formazan crystals were dissolved with an equal volume of solubilization solution, and light absorbance was spectrophotometrically measured at 570 nm. Cell viability for untreated control cultures was set at 100% (dotted line). For each ganciclovir-treated cell culture, the relative cell viability was calculated as follows: (cell viability for that culture/100) × 100%. *Mean differs significantly (P ≤ 0.05) from 100%.

  • Figure 2—

    Mean ± SD percentage of CHV-1–infected MDCK cell culture replicates that contained cytopathic effect following incubation with ganciclovir at serial 2-fold dilutions that ranged from 0 to 800μM. There were at least 3 replicates for each ganciclovir dilution. The CHV-1–infected cells were incubated with the assigned ganciclovir dilution for approximately 72 hours or until cytopathic effect was visible in control cells that were not treated with ganciclovir. The percentage of replicates that contained cytopathic effect was determined for each ganciclovir dilution. The solid line represents the line of best fit for the data and was used to determine the EC50 of ganciclovir for CHV-1 (dotted lines), which was 37.7 μM for this data set.

  • Figure 3—

    Mean ± SD cumulative ocular clinical disease score over time for adult dogs with experimentally induced recurrent ocular CHV-1 infection that, beginning on day 4, received 1 drop of 0.15% ganciclovir ophthalmic ointment gel (ganciclovir group; n = 5; gray bars) or 1 drop of artificial tear ophthalmic ointment gel (control group; 5; black bars) in each eye 5 times daily (at approx 3-hour intervals) for 7 days (days 4 through 10) and then 3 times daily (at approx 6-hour intervals) for another 7 days (days 11 through 17). Beginning on day 1, dogs with latent CHV-1 infection were treated with prednisolone (3.0 mg/kg, PO, q 24 h) for 7 days to make the infection recrudesce. At 2-day intervals throughout the 30-day study period, the severity of blepharospasm, ocular discharge, conjunctival hyperemia, chemosis, conjunctival ulceration, and corneal epithelial ulceration was assessed on a 4-point scale. For all variables except conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = mild, 2 = moderate, and 3 = severe. For conjunctival ulceration and corneal epithelial ulceration, 0 = none, 1 = punctate ulcerations, 2 = ≥ 1 linear or dendritic ulcerations, and 3 = geographic ulcerations. For each dog, a single cumulative ocular clinical disease score was calculated at each evaluation. For dogs that had nonsymmetric ocular disease on any given evaluation, the highest score was used for statistical analyses.

  • Figure 4—

    Representative in vivo confocal photomicrographs of the bulbar conjunctival epithelium (A and B) and corneal basal epithelium (C and D) obtained on day 10 for various dogs in the ganciclovir (B and D) and control (A and C) groups described in Figure 3. Leukocytes infiltrating the tissue appear as highly reflective and irregularly shaped cells. Leukocyte infiltration was considered moderate for dogs in the control group and mild for dogs in the ganciclovir group. Bar = 50 μm. See Figure 3 for remainder of key.

  • 1. Rémond M, Sheldrick P, Lebreton F, et al. Gene organization in the UL region and inverted repeats of the canine herpesvirus genome. J Gen Virol 1996;77:3748.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Krogenæs A, Rootwelt V, Larsen S, et al. A serologic study of canine herpes virus-1 infection in the Norwegian adult dog population. Theriogenology 2012;78:153158.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Babaei H, Akhtardanesh B, Ghanbarpour R, et al. Serological evidence of canine herpesvirus-1 in dogs of Kerman city, south-east of Iran. Transbound Emerg Dis 2010;57:348351.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Carmichael LE, Squire RA, Krook L. Clinical and pathologic features of a fatal viral disease of newborn pups. Am J Vet Res 1965;26:803814.

    • Search Google Scholar
    • Export Citation
  • 5. De Palma VE, Ayala MA, Gobello C, et al. An atypical clinical presentation for the first isolation of canid herpesvirus 1 in Argentina. Arq Bras Med Vet Zootec 2010;62:12671270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Hill H, Maré CJ. Genital disease in dogs caused by canine herpesvirus. Am J Vet Res 1974;35:669672.

  • 7. Karpas A, Garcia FG, Calvo F, et al. Experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally infected dogs. Am J Vet Res 1968;29:12511257.

    • Search Google Scholar
    • Export Citation
  • 8. Ledbetter EC, Kim SG, Dubovi EJ. Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony. Vet Ophthalmol 2009;12:242247.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Ledbetter EC. Canine herpesvirus-1 ocular diseases of mature dogs. N Z Vet J 2013;61:193201.

  • 10. Crumpacker CS. Ganciclovir. N Engl J Med 1996;335:721729.

  • 11. Sahin A, Hamrah P. Acute herpetic keratitis: what is the role for ganciclovir ophthalmic gel? Ophthalmol Eye Dis 2012;4:2334.

  • 12. Matthews T, Boehme R. Antiviral activity and mechanism of action of ganciclovir. Rev Infect Dis 1988;10(suppl 3):S490S494.

  • 13. Field AK, Davies ME, DeWitt C, et al. 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci U S A 1983;80:41394143.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Huang J, Kadonosono K, Uchio E. Antiadenoviral effects of ganciclovir in types inducing keratoconjunctivitis by quantitative polymerase chain reaction methods. Clin Ophthalmol 2014;8:315320.

    • Search Google Scholar
    • Export Citation
  • 15. Kotton CN. CMV: prevention, diagnosis and therapy. Am J Transplant 2013;13(suppl 3):2440.

  • 16. Croxtall JD. Ganciclovir ophthalmic gel 0.15%: in acute herpetic keratitis (dendritic ulcers). Drugs 2011;71:603610.

  • 17. Tabbara KF, Al Balushi N. Topical ganciclovir in the treatment of acute herpetic keratitis. Clin Ophthalmol 2010;4:905912.

  • 18. Castela N, Vermerie N, Chast F, et al. Ganciclovir ophthalmic gel in herpes simplex virus rabbit keratitis: intraocular penetration and efficacy. J Ocul Pharmacol 1994;10:439451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Kaufman HE, Haw WH. Ganciclovir ophthalmic gel 0.15%: safety and efficacy of a new treatment for herpes simplex keratitis. Curr Eye Res 2012;37:654660.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Aggarwal S, Cavalcanti BM, Pavan-Langston D. Treatment of pseudodendrites in herpes zoster ophthalmicus with topical ganciclovir 0.15% gel. Cornea 2014;33:109113.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Koizumi N, Miyazaki D, Inoue T, et al. The effect of topical application of 0.15% ganciclovir gel on cytomegalovirus corneal endotheliitis. Br J Ophthalmol 2017;101:114119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Wong JX, Agrawal R, Wong EP, et al. Efficacy and safety of topical ganciclovir in the management of cytomegalovirus (CMV)-related anterior uveitis. J Ophthalmic Inflamm Infect 2016; 6:10.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Yokogawa H, Kobayashi A, Yamazaki N, et al. Identification of cytomegalovirus and human herpesvirus-6 DNA in a patient with corneal endotheliitis. Jpn J Ophthalmol 2013;57:185190.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Yabiku ST, Yabiku MM, Bottós KM, et al. Ganciclovir 0.15% ophthalmic gel in the treatment of adenovirus keratoconjunctivitis. Arq Bras Oftalmol 2011;74:417421.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Ozen S, Ozer MA. Ganciclovir ophthalmic gel treatment shortens the recovery time and prevents complications in the adenoviral eye infection. Int Ophthalmol 2017;37:245249.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Association for Research in Vision and Ophthalmology. Statement for the use of animals in ophthalmic and vision research. Available at: www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-visual-research/. Accessed Jan 9, 2018.

    • Search Google Scholar
    • Export Citation
  • 27. Ledbetter EC, Spertus CB, Pennington MR, et al. In vitro and in vivo evaluation of cidofovir as a topical ophthalmic antiviral for ocular canine herpesvirus-1 infections in dogs. J Ocul Pharmacol Ther 2015;31:642649.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Ledbetter EC, Dubovi EJ, Kim SG, et al. Experimental primary ocular canine herpesvirus-1 infection in adult dogs. Am J Vet Res 2009;70:513521.

  • 29. Ledbetter EC, da Silva EC, Kim SG, et al. Frequency of spontaneous canine herpesvirus-1 reactivation and ocular viral shedding in latently infected dogs and canine herpesvirus-1 reactivation and ocular viral shedding induced by topical administration of cyclosporine and systemic administration of corticosteroids. Am J Vet Res 2012;73:10791084.

    • Search Google Scholar
    • Export Citation
  • 30. Thomasy SM, Maggs DJ. A review of antiviral drugs and other compounds with activity against feline herpesvirus type 1. Vet Ophthalmol 2016;19 (suppl 1):119130.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Ledbetter EC, Riis RC, Kern TJ, et al. Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs. J Am Vet Med Assoc 2006;229:376384.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Gervais KJ, Pirie CG, Ledbetter EC, et al. Acute primary canine herpesvirus-1 dendritic ulcerative keratitis in an adult dog. Vet Ophthalmol 2012;15:133138.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Spertus CB, Mohammed HO, Ledbetter EC. Effects of topical ocular application of 1% trifluridine ophthalmic solution in dogs with experimentally induced recurrent ocular canine herpesvirus-1 infection. Am J Vet Res 2016;77:11401147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Hobden JA, Kumar M, Kaufman HE, et al. In vitro synergism of trifluorothymidine and ganciclovir against HSV-1. Invest Ophthalmol Vis Sci 2011;52:830833.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Trousdale MD, Nesburn AB, Willey DE, et al. Efficacy of BW759 (9-[[2-hydroxy-1(hydroxymethyl)ethoxy]methyl] guanine) against herpes simplex virus type 1 keratitis in rabbits. Curr Eye Res 1984;3:10071015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Shiota H, Naito T, Mimura Y. Anti-herpes simplex virus (HSV) effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea. Curr Eye Res 1987;6:241245.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Smith KO, Hodges SL, Kennell WL, et al. Experimental ocular herpetic infections in rabbits. Treatment with 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine. Arch Ophthalmol 1984;102:778781.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Colin J, Hoh HB, Easty DL, et al. Ganciclovir ophthalmic gel (Virgan; 0.15%) in the treatment of herpes simplex keratitis. Cornea 1997;16:393399.

    • Search Google Scholar
    • Export Citation
  • 39. Chou TY, Hong BY. Ganciclovir ophthalmic gel 0.15% for the treatment of acute herpetic keratitis: background, effectiveness, tolerability, safety, and future applications. Ther Clin Risk Manag 2014;10:665681.

    • Search Google Scholar
    • Export Citation
  • 40. Wilhelmus KR. Antiviral treatment and other therapeutic interventions for herpes simplex virus epithelial keratitis. Cochrane Database Syst Rev 2015;1:CD002898.

    • Search Google Scholar
    • Export Citation
  • 41. Tsatsos M, MacGregor C, Athanasiadis I, et al. Herpes simplex virus keratitis: an update of the pathogenesis and current treatment with oral and topical antiviral agents. Clin Experiment Ophthalmol 2016;44:824837.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Maggs DJ, Clarke HE. In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. Am J Vet Res 2004;65:399403.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Garré B, van der Meulen K, Nugent J, et al. In vitro susceptibility of six isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet. Vet Microbiol 2007;122:4351.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Smith KO, Galloway KS, Hodges SL, et al. Sensitivity of equine herpesviruses 1 and 3 in vitro to a new nucleoside analogue, 9-[[2-hydroxy-1-(hydroxymethyl) ethoxy] methyl] guanine. Am J Vet Res 1983;44:10321035.

    • Search Google Scholar
    • Export Citation

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