In Vitro and In Vivo Evaluation of Cidofovir as a Topical Ophthalmic Antiviral for Ocular Canine Herpesvirus-1 Infections in Dogs.

PURPOSE: The effects of cidofovir were investigated against canine herpesvirus-1 (CHV-1) in vitro and in dogs with experimentally induced recurrent ocular CHV-1 infection, a host-adapted pathogen animal model of ocular herpes simplex virus-1 (HSV-1) infection. METHODS: The cidofovir EC50 was determined for CHV-1 and HSV-1. A randomized, masked vehicle-controlled trial was performed using beagles with experimentally induced recurrent ocular CHV-1 infection. Dogs received 1 drop of 0.5% cidofovir solution or 0.9% sodium chloride solution (vehicle) in both eyes 2 times daily for 14 days. Dogs were monitored at intervals for 30 days by a clinical ophthalmic examination, in vivo confocal microscopy of the cornea and conjunctiva, ocular sample CHV-1 polymerase chain reaction assay, hemogram, and serum biochemistry panel. Clinical ocular disease scores were calculated and infiltrating leukocytes detected by in vivo confocal microscopy quantified. RESULTS: Cidofovir displayed similar in vitro antiviral activity against CHV-1 and HSV-1. Clinical ocular disease scores were significantly higher in the cidofovir group compared to the vehicle group. Marked conjunctival pigmentation and blepharitis were also detected in the cidofovir group, but not the vehicle group. Conjunctival and corneal leukocyte infiltration scores determined by in vivo confocal microscopy were significantly higher in the cidofovir group. Dogs administered cidofovir had significantly reduced durations of ocular viral shedding compared to the vehicle group. Hemogram and serum biochemistry panel values were unremarkable. CONCLUSIONS: Twice-daily application of topical 0.5% cidofovir ophthalmic solution reduced the duration of ocular viral shedding in dogs with experimentally induced recurrent ocular CHV-1 infection, but was associated with local ocular toxicity.

Mutation hot spots in the canine herpesvirus thymidine kinase gene.

The guanine and cytosine content (GC-content) of alpha-herpesvirus genes are highly variable despite similar genome structures. It is known that drug resistant HSV, which has the genome with a high GC-content (approximately 70%), commonly includes frameshift mutations in homopolymer stretches of guanine (G) and cytosine (C) within the thymidine kinase (TK) gene. However, whether such mutation hotspots exist in the TK gene of canine herpesvirus (CHV) which has a low GC-content was unknown. In this study, we investigated mutations in the TK gene of CHV. CHV was passaged in the presence of iodo-deoxyuridine (IDU), and IDU-resistant clones were isolated. In all IDU-resistant virus clones, mutations in the TK gene were observed. The majority of these mutations were frameshift mutations of an adenine (A) insertion or deletion within either of 2 stretches of eight A's in the TK gene. It was demonstrated that CHV TK mutations frequently occur at a limited number of hot spots within long homopolymer nucleotide stretches.

Corticosteroid treatment does not reactivate canine herpesvirus in red foxes.

To study canine herpesvirus (CHV) reactivation from red foxes (Vulpes vulpes), 29 foxes with varying CHV antibody and CHV carrier status were treated with methylprednisolone acetate, a glucocorticosteroid drug with prolonged immunosuppressive effect in dogs. In the first experiment, 17 foxes with unknown CHV carrier status were treated once with methylprednisolone: in the second experiment, five foxes were treated twice, 4 mo after being intravenously CHV infected; and in the third experiment, six foxes were treated five times, 11 mo after peroral CHV infection. Infectious CHV was not isolated after treatment from either naturally or experimentally CHV-infected foxes or from untreated, CHV-seronegative in-contact foxes. Canine herpesvirus DNA was not detectable in mucosal secretions or white blood cells of any of the foxes, whereas all trigeminal ganglia of experimentally CHV-infected foxes were polymerase chain reaction-positive. In CHV-seropositive foxes, anti-CHV antibody titers did not change with time after treatment, and CHV-seronegative in-contact controls did not seroconvert. Hematologic parameters remained mostly unchanged. We conclude that CHV is not as easily reactivated in foxes following corticosteroid treatment as in dogs, although there was no obvious sign of immunosuppression. Canine herpesvirus was not spread from virus carriers to naive in-contact foxes, which may be among possible explanations for the reported low CHV prevalence in wild foxes.

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.

Attachment and penetration of canine herpesvirus 1 in non-permissive cells.

Canine herpesvirus 1 (CHV-1) has a relatively narrow host cell range when compared to other alphaherpesviruses. The early events of CHV-1 infection in a permissive Madin-Darby canine kidney (MDCK) and non-permissive cell lines. In order to quantify attachment and penetration, were investigated quantitative competitive PCR (QCPCR) method was established for quantitation of CHV-1 DNA. In all non-permissive cells tested, no significant decrease in viral attachment was observed. When CHV-1 was treated with heparin, viral attachment to MDCK cells was reduced by 25% of the input CHV-1 attached to MDCK cells even in the presence of 50 microg/ml heparin. However, the attachment of CHV-1 to non-permissive cells was severely impaired by heparin treatment. In permissive MDCK cells, about 80% of attached CHV-1 penetrated into cells. However, only 4-10% of CHV-1 attached to non-permissive cells penetrated into cells. Our data indicated that CHV-1, like other herpesviruses, attached to permissive MDCK cells through two mechanisms: the first one is through the interaction mediated by heparan sulfate (HS) on the cell surface and the second involves unidentified viral component and the cellular receptor. In contrast, the non-permissive cells lacked the cellular receptor for the second attachment mechanism and the defect in viral penetration into non-permissive cell might be related to the lack of the cellular receptor.

Virus reactivation in bitches with a medical history of herpesvirus infection.

Virologic and pathologic investigations were done on prednisolone-treated bitches with a history of canine herpesvirus (CHV) infection. Reactivation of CHV was demonstrated in 5 Beagle bitches after daily administration of 600 mg of prednisolone for 5 days. The reactivation was confirmed in 4 of 5 bitches. Canine herpesvirus was recovered from nasal, oral, vaginal, and ocular secretions on the 5th to 21st days after initiation of treatment with prednisolone, and also from nasal mucosa and tonsil tissues. Results indicated that latent CHV infections develop and that the virus may be reactivated, without clinical signs, in dogs with a history of CHV infection.