Intensive ocular sampling for the detection of subclinical canine herpesvirus-1 shedding in dogs with experimentally-induced latent infection.

Latent canine herpesvirus-1 (CaHV-1) infections are common in domestic dogs, but viral shedding patterns in dogs are poorly understood. Previous research failed to detect spontaneous subclinical ocular CaHV-1 shedding in dogs following ocular infection, a situation that is fundamentally distinct from many of the alphaherpesviruses closely related to CaHV-1. One possible explanation for this finding is that the sampling interval in the prior studies evaluating ocular shedding patterns was too infrequent to detect rapidly cleared, brief ocular viral shedding episodes. To evaluate for this potential viral shedding scenario, 10 laboratory beagles recovered from experimental primary ocular CaHV-1 infection and with latent CaHV-1infection were intensively monitored for viral reactivation and shedding for 28 days. Clinical ophthalmic examinations were performed daily. Ocular swab samples were collected for CaHV-1 polymerase chain reaction 3 times daily and CaHV-1 virus neutralizing antibody assays were evaluated at 2-week intervals. No abnormalities suggestive of recurrent CaHV-1 ocular disease were observed during clinical ophthalmic examination in the dogs during the study. Ocular CaHV-1 shedding was not detected by polymerase chain reaction and CaHV-1 virus neutralizing antibody titers remained stable in all dogs for the study duration. In the present study utilizing frequent multiple daily sample collections, no evidence of subclinical ocular CaHV-1 shedding was detected in mature dogs with experimentally-induced latent CaHV-1 infection.

New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems.

Ocular herpesviruses, most notably human alphaherpesvirus 1 (HSV-1), canid alphaherpesvirus 1 (CHV-1) and felid alphaherpesvirus 1 (FHV-1), infect and cause severe disease that may lead to blindness. CHV-1 and FHV-1 have a pathogenesis and induce clinical disease in their hosts that is similar to HSV-1 ocular infections in humans, suggesting that infection of dogs and cats with CHV-1 and FHV-1, respectively, can be used as a comparative natural host model of herpesvirus-induced ocular disease. In this review, we discuss both strengths and limitations of the various available model systems to study ocular herpesvirus infection, with a focus on the use of these non-traditional virus-natural host models. Recent work has demonstrated the robustness and reproducibility of experimental ocular herpesvirus infections in dogs and cats, and, therefore, these non-traditional models can provide additional insights into the pathogenesis of ocular herpesvirus infections.

Early events of canine herpesvirus 1 infections in canine respiratory and genital mucosae by the use of ex vivo models.

Canine herpesvirus 1 (CaHV-1) causes a systemic disease in newborn puppies, kennel cough at all ages and genital lesions in adult dogs. The aim of the present study was to elucidate the viral behavior during the early stage of infection in respiratory and genital mucosae, the portals of entry for CaHV-1 by the use of ex vivo explants. CaHV-1 infected and replicated in respiratory and vaginal mucosae in a plaque wise manner. CaHV-1 started to penetrate the basement membrane (BM) only after 48 h post inoculation (hpi) in respiratory mucosal explants, but already after 24 hpi in vaginal explants. The plaque latitude and penetration depth increased over time and both were larger in the vaginal explants compared to the respiratory mucosal explants. The canine respiratory and genital mucosal explants were suitable to study the early pathogenesis of CaHV-1. CaHV-1 showed a better capacity to replicate and invade vaginal mucosa compared to respiratory mucosa, based on the latitude and penetration depth of the plaques of viral antigen positive cells.

Effects of ocular surface strontium-90 beta radiotherapy in dogs latently infected with canine herpesvirus-1.

Latent canine herpesvirus-1 (CHV-1) infections are common in domestic dogs, but stimuli causing viral reactivation and recrudescent disease are poorly understood. Immunosuppressive pharmaceuticals are currently the only experimentally established triggers for recurrent ocular CHV-1 infection in dogs; however, ocular CHV-1 shedding has been reported clinically following strontium-90 beta radiotherapy of the ocular surface and it has been speculated that radiotherapy can directly induce viral reactivation. Strontium-90 is used as a beta radiation source for the treatment of a variety of neoplastic and immune-mediated canine ocular surface diseases. In the present study, the effects of ocular surface strontium-90 beta radiotherapy in dogs latently infected with CHV-1 were evaluated. Ten mature dogs with experimentally induced latent CHV-1 infections were randomly divided into two groups: one group received a single fraction 50 Gy radiation dose in one application from a strontium-90 ophthalmic applicator and the second group received sham radiotherapy. Dogs were then monitored for 45 days for recurrent ocular CHV-1 infection using clinical and virological outcome measures. Clinical ophthalmic examinations, ocular sample CHV-1 PCR assays, and serum CHV-1 virus neutralizing antibody assays were performed at specified intervals. No abnormalities suggestive of recurrent CHV-1 ocular disease were observed on clinical examination in any dog during the study. Ocular viral shedding was not detected and CHV-1 virus neutralizing titers remained stable in all dogs. A single fraction 50 Gy radiation dose administered to the ocular surface by strontium-90 beta radiotherapy did not result in detectable recurrent ocular CHV-1 infection in mature dogs with experimentally induced latent infection.

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.

OBJECTIVE: To determine the frequency of spontaneous canine herpesvirus-1 (CHV-1) reactivation and ocular viral shedding in latently infected dogs and the effect of topical ocular administration of cyclosporine. ANIMALS: 8 mature Beagles with experimentally induced latent CHV-1 infection. PROCEDURES: Following induction of primary ocular CHV-1 infection, the presence of reactivatable CHV-1 latency was confirmed by systemically administering prednisolone to the dogs. Dogs were then monitored for 36 weeks via clinical examination and conjunctival sample CHV-1 PCR assay performed at 4-day intervals and CHV-1 virus neutralization antibody assay performed at 2-week intervals. During weeks 16 to 32, dogs were administered 0.2% cyclosporine ointment in both eyes twice daily and blood cyclosporine concentrations were monitored. During weeks 33 to 36, the presence of reactivatable CHV-1 latency was reconfirmed via systemic administration of prednisolone. RESULTS: Reactivation of latent CHV-1 was not detected via clinical examination or viral shedding during the initial 32 weeks, including before and during topical ocular administration of cyclosporine, and there were no significant differences in CHV-1 virus neutralization titer increases between the study periods. Blood cyclosporine concentrations were less than assay detection limits in all dogs on the sampling days. Systemic administration of corticosteroids repeatedly resulted in ocular disease and viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE: Spontaneous CHV-1 reactivation did not occur frequently in latently infected mature dogs, and this was not altered by topical ocular administration of cyclosporine. This characteristic may be a factor contributing to the lower frequency of recurrent herpetic ocular disease in dogs relative to other host species and their associated alphaherpesviruses.

Effects of cyclophosphamide myelosuppression in adult dogs with latent canine herpesvirus-1 infection.

Latent canine herpesvirus-1 (CHV-1) infection is common in domestic dogs, but triggers for viral reactivation and recrudescent CHV-1 disease are poorly understood. Cyclophosphamide is a potent immunosuppressive and myelosuppressive agent used for the therapy of a variety of neoplastic and immune-mediated canine disorders. Cyclophosphamide (200mg/m(2)) was administered to mature dogs latently infected with CHV-1 to determine its potential to induce recurrent CHV-1 disease and viral shedding. Non-infected dogs and dogs recovered from experimental primary ocular CHV-1 infection with experimentally confirmed latent CHV-1 infection were divided into groups and administered cyclophosphamide or placebo. Dogs were monitored for myelosuppression and viral reactivation for 28days using clinical and virological outcome measures. Clinical ophthalmic and in vivo ocular confocal microscopic examinations were performed at intervals. Samples were collected for CHV-1 polymerase chain reaction (PCR), CHV-1 virus neutralizing (VN) antibody, and hemogram assays. Myelosuppression (i.e., decreased total leukocyte, segmented neutrophil, and erythrocyte counts) was detected on study day 7 in dogs administered cyclophosphamide, but not dogs administered placebo. There were no abnormalities suggestive of recurrent CHV-1 ocular disease during clinical ophthalmic or in vivo confocal microscopic examination in any dogs during the study. Ocular CHV-1 shedding was not detected by PCR and CHV-1 VN titers remained stable in all dogs. Following study conclusion, the presence of reactivatable latency was reconfirmed in the infected dogs by administering systemic prednisolone. Myelosuppression elicited by a single dose of cyclophosphamide does not result in detectable recurrent ocular CHV-1 infection in adult dogs with experimentally induced latent CHV-1 infection.

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

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

Experimental primary ocular canine herpesvirus-1 infection in adult dogs.

OBJECTIVE-To characterize clinical ocular disease, viral shedding, and serologic response associated with primary canine herpesvirus-1 (CHV-1) ocular infection in naïve adult dogs. ANIMALS-12 specific pathogen-free adult Beagles. PROCEDURES-Dogs were topically inoculated in the right eye with CHV-1 (infection group; n = 8) or virus-free medium (control group; 4). Dogs were inoculated with or without corneal microtrephination and subconjunctivally administered corticosteroids. Conjunctiva, buffy coat, and serum samples for real-time PCR assay, virus isolation, and serum neutralization (SN) antibody titers were collected until postinfection day (PID) 224, and general physical and ophthalmologic examinations were performed. RESULTS-Dogs in the infection group developed bilateral, mild to moderate conjunctivitis that reached maximal intensity on PIDs 7 to 10. Ocular viral shedding was detected in all dogs in the infection group between PIDs 3 and 10. Infected dogs developed CHV-1 SN antibody titers, beginning at PID 7 and peaking on PID 21. All buffy coat PCR assay results were negative. Corneal microtrephination and subconjunctival corticosteroid administration did not significantly affect clinical disease or viral shedding. Following recovery from primary infection, dogs remained clinically normal, did not shed virus, and had slowly decreasing SN antibody titers. Dogs in the control group did not develop conjunctivitis, shed virus, or develop CHV-1 SN antibody titers. CONCLUSIONS AND CLINICAL RELEVANCE-Primary ocular infection of adult dogs with CHV-1 was associated with self-limiting conjunctivitis and ocular viral shedding, which was evident in the absence of clinically detectable keratitis or systemic disease. Features of this infection resembled herpes simplex virus primary ocular infection in humans.

Experimental reactivation of latent canine herpesvirus-1 and induction of recurrent ocular disease in adult dogs.

Latent canine herpesvirus-1 (CHV-1) infection is common in domestic dogs, but recrudescent CHV-1 diseases are poorly characterized. To determine if administration of an immunosuppressive dosage of prednisolone to adult dogs latently infected with CHV-1 results in recurrent ocular disease, adult beagles with and without experimentally induced CHV-1 latent infection were divided into groups: group 1 latently infected and administered prednisolone, group 2 latently infected and administered placebo, and group 3 not latently infected and administered prednisolone. Prednisolone (3.0 mg/kg/day) was administered to dogs in groups 1 and 3 for seven consecutive days beginning on study day 1. Samples for CHV-1 polymerase chain reaction and serum neutralization (SN) assays were collected, and physical, ophthalmologic, and in vivo ocular confocal microscopic examinations were performed at intervals for 42 days. Bilateral ocular disease (i.e., conjunctivitis or keratitis) was detected in 83% of group 1 dogs between study days 3 and 18. In vivo confocal microscopic abnormalities included conjunctival leukocyte infiltration and corneal leukocyte infiltration, abnormal epithelial cell morphology, and Langerhans cell infiltration. Ocular viral shedding was detected in 50% of group 1 dogs on study days 10 and 13. Fourfold elevations in CHV-1 SN titers were detected in 100% of group 1 dogs by study day 14. Dogs in control groups did not develop clinical ocular disease (P<0.05), CHV-1 titer elevations (P<0.005), or viral shedding. Administration of an immunosuppressive dosage of systemic prednisolone to adult dogs latently infected with CHV-1 may result in viral reactivation and ocular disease recrudescence.

The replication of canine herpesvirus (CHV) induces apoptosis in canine kidney cell line: short communication.

The alphaherpesvirus canine herpesvirus (CHV) was tested in order to determine whether or not it has apoptotic potential. We have demonstrated that lytic replication of CHV resulted in induction of apoptosis. This phenomenon was confirmed using different techniques including in situ TUNEL assay and DNA laddering. The apoptotic activity of CHV might influence the pathobiology of this virus.

Antiviral activity of lactoferrin against canine herpesvirus.

Lactoferrin (LF) is an iron-binding protein that is found in milk and other mammalian secretions. We found that bovine lactoferrin (bLF) inhibited both the in vitro infection and replication of canine herpesvirus (CHV) in Madin-Darby canine kidney (MDCK) cells. Incubation of CHV with bLF prevented subsequent infection of MDCK cells. Furthermore, proteins from CHV-infected MDCK cells were resolved by SDS-PAGE, and then bLF CHV-binding proteins were identified by far Western blotting. We demonstrated that the anti-CHV activity of bLF was due to its interaction with CHV as well as with MDCK cells. Both the apo- and holo-forms of bLF inhibited virus multiplication independently of their iron-withholding properties. We also demonstrated that human LF had anti-CHV activity. Our findings suggest that LF could be effective in dogs to provide protection against CHV infection.