Canine herpesvirus-1 is a varicellovirus of the subfamily Alphaherpesvirinae with a worldwide distribution and host range restricted to canids.1 During primary CHV-1 infection, virus is transported in sensory nerves by retrograde axonal transport to regional sensory ganglia, where it establishes latency.2 Depending on the specific canine population evaluated, 39% to 93% of dogs are latently infected with CHV-1.3–8 Historically, CHV-1 was primarily regarded as a pathogen of neonates; however, recent studies9–12 and clinical reports have established the etiologic importance of CHV-1 as an ocular, genital, and respiratory pathogen of mature dogs. In contrast to neonates, CHV-1 disease in mature dogs may be associated with primary or recurrent infection. During CHV-1 reactivation, anterograde neuronal transport of virus back to the peripheral site of primary infection occurs and is associated with viral replication, viral shedding, and recurrent disease.13–15 Ocular lesions in mature dogs associated with primary and recurrent CHV-1 infection include conjunctivitis; punctate, dendritic, and geographic ulcerative keratitis; and nonulcerative keratitis.9,10,16
Features of CHV-1 latency, including reactivation frequency and induction stimuli, have not been established. Systemic administration of immunosuppressive corticosteroids, but not topical ocular administration of corticosteroids, induces latent CHV-1 reactivation in mature dogs following natural and experimental infection.13–15,17 Reactivation of latent CHV-1 in experimental studies13–15 is associated with mucosal viral shedding and can be subclinical or associated with recrudescent disease. Some alphaherpesviruses, including HSV and FHV-1, have periodic viral reactivation and mucosal viral shedding at the infected end organ in their natural hosts, with or without overt recurrent clinical disease, in the absence of identifiable reactivation stimuli or immunocompromise.18–21 The occurrence and frequency of spontaneous CHV-1 reactivation in immunocompetent mature dogs have not been reported.
Cyclosporine is an immunosuppressive agent that blocks recruitment and activation of T lymphocytes.22 Topical ophthalmic cyclosporine formulations are commonly administered to dogs for treatment of immune-mediated and inflammatory ocular conditions, including keratoconjunctivitis sicca and chronic superficial keratitis.23–29 Following topical ocular administration in dogs, cyclosporine is concentrated in ocular tissues and distributed systemically, where it may suppress peripheral lymphocyte function.30–32 Current evidence suggests there is a dynamic interaction between the host immune system and latent alphaherpesvirus infection, where acquired immunity actively controls virus latency and prevents viral reactivation.33 It is proposed that host T lymphocytes play a prominent role in controlling alphaherpesvirus latency and blocking early steps of viral reactivation, a function that could be interrupted by immunosuppressive agents like cyclosporine.34–36 Recurrent clinical CHV-1 ocular disease has been reported in dogs during topical ocular administration of cyclosporine, but the contribution of cyclosporine to viral reactivation is not clear.16
The high prevalence of latent CHV-1 infection and frequent use of topical ocular administration of cyclosporine in mature dogs render this potential virus-pharmaceutical interaction of clinical importance. Many of the canine ocular conditions for which cyclosporine is administered topically to the eyes are associated with lesions clinically indistinguishable from recurrent CHV-1 ocular infection, and therapeutic decisions are frequently made solely on the basis of clinical observations.9,10,23–29 Therefore, the objectives of the study reported here were to determine the frequency of spontaneous CHV-1 reactivation and ocular viral shedding in latently infected mature dogs and to evaluate the effects of topical ocular administration of cyclosporine on these variables.
Materials and Methods
Animals, study design, and study agents—All protocols were approved by the Animal Care and Use Committee of Cornell University 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. Eight specific pathogen–free 3-year-old sexually intact male Beagles with experimentally induced CHV-1 latent infection were used. Dogs were maintained individually in runs separated by 1.2 m, and direct contact between dogs was prevented for the duration of the study. Strict bio-isolation was maintained throughout the study for all personnel in contact with dogs. Dogs were acclimated to housing facilities for a minimum of 12 weeks prior to the beginning of the study.
Latent CHV-1 infection was experimentally induced by topical ocular inoculation, with or without corneal microtrephination and subconjunctival corticosteroids, 26 months prior to the beginning of the present study, as described.37 Briefly, naïve dogs were topically inoculated in the right eye with 2 × 105 TCID50 of a field strain of CHV-1 isolated from corneal samples of a dog with dendritic ulcerative keratitis treated at the Cornell University College of Veterinary Medicine Hospital for Animals, Ithaca, NY. Eight months after recovery from primary ocular infection, the presence of reactivatable CHV-1 latency was experimentally confirmed as described13 via systemic administration of an immunosuppressive dose of prednisolone (3.0 mg/kg, PO, q 24 h) to the dogs for 7 consecutive days. The dogs were used in a study17 evaluating the effects of topical ocular administration of corticosteroids on viral latency 12 months following viral reactivation induced with systemic administration of prednisolone; however, CHV-1 reactivation was not detected during the course of the study. Primary and recurrent ocular CHV-1 infections in the dogs were evaluated via clinical examination, CHV-1 VN antibody titer, conjunctival swab specimen virus isolation, and conjunctival swab specimen CHV-1 PCR assay. The dogs were maintained in isolation facilities until the present study.
Total study duration was 36 weeks, divided into 3 investigative periods. During weeks 1 to 16, the frequency of apparently spontaneous CHV-1 reactivation was evaluated without additional experimental manipulation of the dogs. During weeks 17 to 32, the frequency of CHV-1 reactivation was evaluated during topical ocular treatment with cyclosporine and each dog was administered a 1/4-inch strip of a commercially manufactured 0.2% cyclosporine ointmenta in both eyes at 8:00 am and 8:00 pm daily. During weeks 33 to 36, the presence of reactivatable CHV-1 latency in the dogs was reconfirmed by administering prednisoloneb (3.0 mg/kg, PO, q 24 h) for 7 consecutive days. Cyclosporine ointment was not administered during weeks 33 to 36. In all study periods, the presence of CHV-1 reactivation was determined by detection of recrudescent ocular disease, serologic response, and viral shedding.
Clinical examination and sample collection—Samples for CBC and serum biochemical analysis were collected from each dog immediately prior to the study. Complete physical and ophthalmic examinations, including slit-lamp biomicroscopy,c indirect ophthalmoscopy, Schirmer I tear testing, and corneal application of lissamine green stain were performed on each dog prior to study initiation. Ophthalmic examination of both eyes with slit-lamp biomicroscopy, before and after application of lissamine green stain, was performed every 4 days during the duration of the study. An ocular surface disease clinical scoring system was used to quantify examination findings. The following ocular variables were scored: blepharospasm, ocular discharge, conjunctival hyperemia, chemosis, and corneal epithelial ulceration. All variables, except corneal epithelial ulceration, were scored as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Corneal epithelial ulceration was scored as follows: 0 = none, 1 = punctate ulcerations, 2 = ≥ 1 linear or dendritic ulceration, and 3 = geographic ulcerations.
All diagnostic assays were performed immediately prior to study initiation and at defined intervals during the study duration. Following initial clinical scoring, but prior to lissamine green application, conjunctival swab specimens were collected from both eyes of each dog every 4 days for CHV-1 PCR assay by brushing sterile polyester-tipped swabsd across the conjunctival fornices. Serum for CHV-1 VN antibody titers was collected every 14 days. Swab and serum samples were stored in sterile tubes at −80°C until analysis. Whole blood with EDTA anticoagulant was collected 1 hour after the 8:00 am cyclosporine ointment application at 28-day intervals during the period of cyclosporine administration and processed immediately for determination of cyclosporine concentrations.
Multiplex real-time PCR assay analysis—Extraction of DNA from ocular swab samples was performed by use of a DNA purification kit.e A multiplex real-time PCR assay targeting the thymidine kinase gene (GenBank accession No. AB038340) and glyceraldehyde-3-phosphate dehydrogenase gene (GenBank accession No. D83054) was used. The primers and probes were designed with a commercial programf as described.13 The primers and probe for the thymidine kinase gene were CHVTK308F 5′-CTG GCG TAT CAT CCT AGA AAC AGA T-3′, CHVTK374R 5′-ACG CAG CTT CCG GTC TTG TA-3′, and CHVTK334P FAM 5′-TTG TGC AAG GTA TAT ACT C-MGB-3′. The primers and probe for the glyceraldehyde-3-phosphate dehydrogenase gene were K9GAPDHF 5′-CCC ACC CCC AAT GTA TCA GT-3′, K9GAPDHR 5′-TGT CGT CAT ATT TGG CAG CTT T-3′, and K9GAPDHP VIC 5′-TGG ATC TGA CCT GCC G-MGB-3′. The PCR assay was performed by use of a real-time PCR system,g with the reaction mixture prepared with a premixed solutionh as described.37 The thermocycler conditions were as follows: activation of DNA polymerase at 95°C for 29 seconds, 40 cycles of denaturation at 95°C for 3 seconds, and primer annealing and extension at 60°C for 30 seconds. The detection limit of the CHV-1 PCR assay was 1 to 10 TCID50/mL.
CHV-1 VN antibody titer—Test sample, high positive control, low positive control, and negative control sera were heat-inactivated in a 56°C water bath for 30 minutes. Test medium consisting of 50 μL of Eagle minimum essential mediumi with heat-inactivated 10% fetal bovine serumj and 2% penicillin-streptomycin solution was added to plate wells along with 50 μL of test sample, and 2-fold serial dilutions were performed. The working dilution of virus (100 TCID50/50 μL) and back titrations of 10−-1, 10−-2, and 10−-3 dilutions were prepared and added to test wells. Plates were incubated for 1.5 hours at 37°C. Canine kidney DE-2 cellsk were added to the wells and incubated for 3 days at 37°C in a humidified 5% CO2 incubator. An inverted microscope was used to examine wells for cytopathic effect, and antibody titers were calculated.
Cyclosporine blood concentrations—Cyclosporine concentrations were determined in whole blood via an automated fluorescent polarization immunoassay.l Standards, provided with the system, were run prior to each set of assays. The limit of detection of the assay was 25 ng of cyclosporine/mL.
Statistical analysis—For data analysis, clinical ocular disease scores, ocular viral shedding, and CHV-1 VN titer elevations were summarized into dichotomous variables. The presence or absence of clinically detectable ocular disease (ie, at least 1 study day's clinical score > 0), ocular viral shedding (ie, detected by CHV-1 PCR assay on at least 1 study day), and ≥ 4-fold increases in CHV-1 VN antibody titers (between samples collected during any 28-day study period on a rolling basis) were compared between the study periods involving no medication (ie, weeks 1 to 16) and topical ocular administration of cyclosporine (ie, weeks 17 to 32) with the Fischer exact test. Statistical calculations were performed with a commercially available software package.m Values of P ≤ 0.05 were considered significant for all comparisons.
Results
No abnormalities were detected in any dog during physical and ophthalmic examinations performed prior to the study. Schirmer I tear test results were > 15 mm/min in both eyes of all study dogs, and corneal retention of lissamine green stain was not observed. Results of CBC and serum biochemical analysis were unremarkable for each dog.
During weeks 1 to 32, including before and during topical ocular administration of cyclosporine, clinical ocular disease was not detected in any dog (ie, all daily clinical scores = 0) on the selected examination days. Results of all conjunctival swab specimen CHV-1 PCR assays were negative for each dog during this study period. The mean CHV-1 VN antibody titer for all study dogs differed among sampling days during weeks 1 to 32, including before and during topical ocular administration of cyclosporine, with a minimum mean ± SD titer of 6.6 ± 3.4 on study day 56 and a maximal mean ± SD titer of 41.4 ± 50.6 on study day 210 (Figure 1). Although mean CHV-1 VN antibody titer values differed among sampling days, a ≥ 4-fold titer increase was only detected over a 28-day period on 5 occasions and in 3 dogs, including 3 dogs during the no-medication study period and 2 dogs during topical ocular administration of cyclosporine. The difference between the frequency of ≥ 4-fold CHV-1 VN antibody titer increases was not significantly (P = 1.0) different between weeks 1 to 16 (no medication) and weeks 17 to 32 (topical administration of cyclosporine). Blood cyclosporine concentrations were less than assay detection limits in all dogs on the selected sampling days.
Mean ± SD CHV-1 VN antibody titers for 8 mature Beagles latently infected with CHV-1. Titers were evaluated at 2-week intervals for 36 weeks. No medications were administered to dogs during study weeks 0 to 16. Cyclosporine was administered topically to the eyes of dogs after study week 16 until study week 32. Prednisolone was administered systemically to dogs for 7 days beginning after study week 32. Titers are presented as the reciprocal of the highest dilution of the serum that neutralized the infectivity of virus and are in units of antibody.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1079
Following systemic administration of prednisolone in the final study month, clinical ocular disease was detected in 7 study dogs. Each of these dogs had bilateral mild-to-moderate conjunctivitis that was characterized by intermittent blepharospasm, conjunctival hyperemia, chemosis, and mucopurulent ocular discharge. No corneal epithelial defects were detected via corneal application of lissamine green. No ocular disease was detected in 1 dog during this period (ie, all daily clinical scores = 0). In dogs that developed ocular disease, the median onset of clinically detectable conjunctivitis was 8 days (range, 4 to 12 days) after initiating prednisolone administration (Figure 2). The mean ± SD duration of detectable ocular disease was 10.0 ± 7.3 days. Ocular CHV-1 shedding was detected in 3 dogs with conjunctivitis during the period of recurrent disease. In each of these 3 dogs, conjunctival swab specimen CHV-1 PCR assays were positive for samples collected both 8 and 12 days after initiating prednisolone administration. The mean ± SD CHV-1 VN antibody titer for all study dogs increased from 9.8 ± 6.7 immediately prior to prednisolone administration to 31.3 ± 32.0 and 51.3 ± 41.5 on days 14 and 28, respectively, after prednisolone administration was initiated (Figure 1). A 4-fold CHV-1 VN antibody titer increase was detected in 6 dogs during the 28-day period after administering prednisolone. Canine herpesvirus-1 VN antibody titers remained stable in the other 2 study dogs after prednisolone administration.
Mean ± SD ocular disease clinical scores for dogs latently infected with CHV-1 and systemically administered prednisolone. Prednisolone was administered for 7 days beginning on study day 224, and clinical scores were calculated every 4 days for 28 days.
Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1079
Discussion
Canine herpesvirus-1 reactivation was not detected during the 36-week study period prior to prednisolone administration. This suggests that CHV-1 does not reactivate spontaneously in immunocompetent mature dogs or that viral reactivations occur at a frequency less than detection limits of the present study. In contrast to CHV-1, apparently spontaneous reactivation of FHV-1 was detected via oropharyngeal and ocular viral shedding in 29% of cats during a similar observation period (mean sampling period, 8.8 months).18 Herpes simplex virus-1 is intermittently shed in tears or saliva at least once per month in 98% of humans, and ocular viral shedding is detected in 35% of individuals at any given time.19 Accurate prevalence estimates for recurrent CHV-1 ocular diseases in dogs are not available; however, on the basis of the relative frequency of descriptions in the literature, recurrent ocular disease associated with FHV-1 and HSV-1 infection appear to be more common in cats and humans, respectively.38,39 Relative host species differences in viral reactivation frequencies may be a major factor contributing to the lower frequency of recurrent herpetic ocular disease in dogs relative to cats and humans.
Reported triggers for alphaherpesvirus recrudescent disease (eg, corticosteroids, fever, hormonal fluctuations, stress, surgery, trauma, and UV light) were historically assumed to stimulate latent virus reactivation, viral shedding, and subsequent disease.40 The high frequency of apparently spontaneous viral reactivation and peripheral shedding that occurs in the absence of clinical disease with FHV-1 and HSV-1 suggests recrudescent peripheral tissue disease only occurs when local immunity is impaired.41 If this theory is correct, recrudescent disease triggers may actually modify the immune response to spontaneous reactivation or viral reactivation induced by a different stimulus, as opposed to directly triggering viral escape from latency.41 In this pathophysiologic scenario, the low frequency of spontaneous CHV-1 reactivations would provide limited opportunities for the development of recurrent herpetic disease in dogs. This explanation does not adequately address the high frequency of viral shedding and recrudescent ocular disease detected in the dogs of the present study during systemic administration of prednisolone, unless peripheral tissue sampling does not always reflect what is occurring at sites of viral latency. The present study evaluated viral reactivation primarily by end organ testing, and it is possible that sampling at sensory ganglia for infectious virus, viral proteins, or viral gene expression could yield different results.42 If CHV-1 reactivation occurred in sensory ganglia during the study, it did not proceed to detectable viral shedding or recrudescent disease except with prednisolone administration. Prednisolone may induce recurrent CHV-1 disease and shedding in dogs by impairing the ability of the canine immune system to prevent viral escape from latency, transport to peripheral tissues, or replication.
Another possible explanation for failure to detect CHV-1 reactivation in the present study is that short, self-limiting reactivations occurred between sampling and examination periods. By use of intensive mucosal sampling for viral detection (ie, 4 times/d), it has been reported that 49% of anogenital HSV-2 reactions and 39% of oral HSV-1 reactions last < 12 hours in humans.20 Mathematical modeling of daily genital shedding patterns in humans has determined that extended reactivation episodes of several days are caused by multiple coinciding HSV-2 reactivations instead of a single viral reactivation.43 The persistence of HSV-specific CD8(+) T lymphocytes in mucosa and skin adjacent to peripheral sensory nerve endings following HSV reactivation suggests that immunocompetent host defense mechanisms provide effective immune surveillance at end organs and are capable of rapidly eliminating reactivated virus at peripheral sites.44 Considering the duration and frequency of sampling in the present study, if rapidly cleared CHV-1 reactivations occurred in study dogs, they would likely be infrequent to completely escape detection in all dogs over the entire observation period.
Twice-daily ocular application of 0.2% cyclosporine ointment did not affect detectable CHV-1 reactivation frequency in the study dogs, suggesting this treatment is safe in dogs with historical CHV-1 ocular disease and no active epithelial lesions. There are contradictory descriptions of the effect of topical ophthalmic administration of cyclosporine on canine peripheral immune function in the clinical setting.32,45 In 1 report,32 twice-daily topical ocular administration of 2% cyclosporine to dogs was associated with suppression of peripheral lymphocyte proliferation that was measured after 1 and 3 months of treatment. More recently, twice-daily topical ocular application of both 0.2% and 2% cyclosporine to dogs was reported to not affect lymphocyte proliferation.45 In humans, topical ocular administration of cyclosporine is clinically associated with persistent HSV-1 keratitis that is refractory to treatment; however, there is presently no evidence this route of administration directly induces viral reactivation.46 Similarly, prolonged episodes of CHV-1 ulcerative keratitis are reported in dogs being treated with topical ocular administration of cyclosporine that were refractory to antiviral treatment but these episodes rapidly resolved following cessation of cyclosporine administration.16 The safety of ophthalmic administration of cyclosporine in dogs with active CHV-1 epithelial disease, concurrent ocular surface disease, or immunomodulating systemic disease was not evaluated during the present study and is presently unknown.
In the present study and a previous study,45 application of 0.2% cyclosporine ointment failed to result in detectable blood cyclosporine concentrations in dogs. Systemic treatment with cyclosporine or topical ocular administration of a higher drug concentration that results in greater systemic drug distribution may yield different findings in dogs with latent CHV-1 infection than did the present study. Systemic administration of cyclosporine increases the severity of clinical lesions, viral shedding, and ocular inflammatory infiltrates in animals with experimental HSV-1 keratitis.47,48 Recurrent HSV infections occur commonly in humans receiving systemic treatment with cyclosporine.49–51 Systemic treatment with immunosuppressive doses of cyclosporine predisposes dogs to a variety of opportunistic infections, including disseminated nocardiosis, toxoplasmosis, and phaeohyphomycosis; however, the risk of recurrent CHV-1 infection is unknown.52–55
In immunocompetent healthy mature dogs latently infected with CHV-1, spontaneous CHV-1 reactivations that proceeded to detectable ocular viral shedding and recrudescent ocular disease occurred infrequently under experimental conditions. This characteristic was not altered by twice-daily topical ocular application of 0.2% cyclosporine ointment.
ABBREVIATIONS
| CHV | Canine herpesvirus |
| FHV | Feline herpesvirus |
| HSV | Herpes simplex virus |
| VN | Virus neutralization |
Optimmune, Schering-Plough Animal Health, NJ.
Lloyd Inc, Shenandoah, Iowa.
Kowa SL-15, Kowa Co, Tokyo, Japan.
Puritan Medical Products Co, Guilford, Me.
DNeasy Blood and Tissue Kit, Qiagen, Valencia, Calif.
Primer Express, version 2, Applied Biosystems, Foster City, Calif.
AB 7500 Fast Real-time Detection System, Applied Biosystems, Foster City, Calif.
Applied Biosystems Fast Universal Master Mix, Applied Biosystems, Foster City, Calif.
Invitrogen Co, Carlsbad, Calif.
Atlantic Biologicals Co, Atlanta, Ga.
New York State Animal Health Diagnostic Center, Ithaca, NY.
TDx Immunosuppressant Drug Assays—Cyclosporine Monoclonal Whole Blood, Abbott Laboratories, Chicago, Ill.
Statistix, version 9, Analytical Software, Tallahassee, Fla.
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