Feline herpesvirus-1 is a varicellovirus of the subfamily Alphaherpesvirinae. It is a double-stranded DNA virus with a short replication cycle that spreads rapidly from cell to cell, tends to induce cell lysis, and persists in the sensory ganglia of the host.1 The virus is ubiquitous in feline populations worldwide, with a reported seroprevalence of up to 97% in domestic cats.2–4 Approximately 80% of FHV-1–infected cats remain latently infected for life, and approximately 45% of latently infected cats will intermittently shed FHV-1 spontaneously or subsequent to some natural stress that reactivates the virus.5 Feline herpesvirus-1 is the most common viral cause of ocular surface disease in cats, the clinical lesions of which include conjunctivitis, acute corneal ulceration, and chronic stromal keratitis.1,4,6 Primary infection with FHV-1 is typically self-limiting, with signs of the disease lasting only a few weeks followed by clinical recovery.6 Feline herpesvirus-1 vaccines are widely used and can decrease the severity of clinical signs, but they confer only partial protection against infection, viral shedding, and latency load.4
The efficacy of several systemic medications against FHV-1 infection in cats has been investigated. Famciclovir is the prodrug of penciclovir and is converted to the active drug following absorption across the gastrointestinal tract mucosa. Although famciclovir is often clinically effective, the pharmacokinetics of penciclovir following oral administration of famciclovir in cats is complex and nonlinear, with significant variability among cats.7–9 Oral administration of interferons and l-lysine to FHV-1–infected cats has yielded equivocal results, and their efficacy remains questionable.1,8
Acyclic nucleoside analogs are the most effective group of anti-herpesvirus drugs for the topical treatment of ocular FHV-1 infection. However, most of those antivirals require frequent application, and many have not been evaluated in well-controlled in vivo studies to justify their use in clinical patients.1,8 Cidofovir is one of the few nucleoside analogs that has been evaluated in cats under controlled experimental conditions. In cats, cidofovir has a long half-life and persists in ocular tissues for an extended period of time, which allows for a fairly long dosing interval. In 1 study,10 cats with experimentally induced ocular FHV-1 infection that received topical ophthalmic application of a 0.5% cidofovir solution twice daily for 10 days had significantly lower clinical scores (ie, less severe clinical signs) and ocular viral shedding than similar cats that received the control treatment.
Although nucleoside analogs are generally effective against herpesviruses, viral mutation can lead to resistance.11,12 Results of a study by Yan et al11 suggest that small-molecule HIV integrase inhibitors might complement treatment with traditional nucleoside analogs and polymerase-helicase inhibitors. Raltegravir is a retroviral integrase inhibitor that inhibits the nuclease activity of the terminase protein of human cytomegalovirus13 and interferes with UL42, the DNA polymerase processivity factor of human alphaherpesvirus HSV-1.14 Feline herpesvirus-1–encoded UL42 shares 24% amino acid sequence similarity with HSV-1 UL42.15
Raltegravir is widely used for treatment of HIV-infected humans16 and appears to have activity against FeLV.17–19 In an in vitro study20 that involved cell culture evaluation and a novel corneal explant model system, a 500μM concentration of raltegravir had no toxic effects on feline cells and corneas and inhibited FHV-1 replication in both systems. In the whole corneal explant model system used in that study,20 raltegravir (500μM) effectively inhibited FHV-1 replication and prevented FHV-1–induced epithelial thinning when administered only once daily. Cats appear to tolerate raltegravir well, and systemic administration of raltegravir at dosages up to 80 mg (ie, 20 to 25 mg/kg) twice daily can be safely administered to cats.19
The purpose of the study reported here was to determine the effects of orally administered raltegravir in cats with experimentally induced ocular and respiratory FHV-1 infection. Specifically, we sought to evaluate the effect of the drug on ocular clinical disease scores, respiratory clinical disease scores, respiratory rate, ocular viral load, ocular viral shedding, and results of in vivo confocal microscopic examination of the cornea.
Materials and Methods
Animals
All study protocols 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.21 Fourteen 6-month-old unvaccinated specific pathogen–free (ie, cats were seronegative for antibodies against FHV-1, FeLV, and FIV) domestic shorthair cats were acquired for the study. The cats had a mean ± SD weight of 3.0 ± 0.4 kg at study initiation. Cats were individually housed in an isolation facility, and direct contact among cats was prohibited for the duration of the study. All personnel who came into contact with the study cats were required to follow a strict biosecurity protocol, which included the use of personal protective equipment such as hazardous material suits, gloves, and boot covers, for the duration of the study. Cats were allowed to acclimate to the housing facilities for 2 weeks before study initiation and were adopted into private households following study completion.
Study design and experimental inoculation of FHV-1
A randomized, masked, 30-day, vehicle-controlled study was performed. On day 0, baseline data were obtained, and all cats were experimentally inoculated with FHV-1 by means of an ocular drop method similar to that used to experimentally inoculate dogs with canine herpesvirus-1.22,23 Briefly, 0.1 mL of a solution containing 106 plaque-forming units of FHV-1 strain FH2CSa was topically applied to the inferior conjunctival fornix of each eye as described.24 Cats were then randomly allocated by means of a random number generator to 1 of 2 groups (ie, block randomization; 7 cats/group). One group was designated to receive raltegravir, and the other was designated to receive lactose powder (vehicle), orally, twice daily for 14 days beginning on day 1. The 2 treatments were compounded in identical capsules so the study administrator and investigators responsible for examining the cats and collecting data remained unaware of (were masked to) the treatment assigned to each cat for the duration of the 30-day observation period.
Study treatments
Forty 400-mg raltegravir tabletsb from the same lot and batch were used to make a single batch of capsules for administration to the cats in the raltegravir group. Each capsule contained 80 mg of raltegravir and sufficient lactose powderc to fill a No. 3 capsule (capacity, 250 mg). The tablets were weighed to determine how much lactose powder (vehicle) would need to be added to achieve the desired raltegravir concentration in the compounded capsules. Then, the tablets were ground into a fine powder by use of a mortar and pestle. The previously calculated amount of lactose powder was added to the raltegravir powder, and the mixture was thoroughly combined. A capsule-filling machine was used to place the compounded mixture into the capsules. Identical capsules were filled with lactose powderc (250 mg/tablet) in the same manner for the cats in the vehicle group. The capsules for each group were placed in identical external containers, which were uniquely labeled so that investigators remained unaware of which treatment was in each container and stored at room temperature (approx 22°C).
Given the body weight of the cats in the raltegravir group at study initiation, it was estimated that the dosage of the drug administered ranged from 23 to 32 mg/kg (mean ± SD, 27.3 ± 3.4 mg/kg), PO, every 12 hours for 14 days. The cats in the vehicle group received lactose (250 mg), PO, every 12 hours for 14 days. All capsules were administered by use of a pet pill dispenser.
Clinical examination and blood sample collection
For each cat, a complete respiratory examination and ophthalmic examination (Schirmer tear test,d slit-lamp biomicroscopye before and after application of fluorescein stain,f applanation tonometry,g and indirect ophthalmoscopyh of both eyes) were performed on days 0 (baseline), 15, and 30. A respiratory examination and abbreviated ophthalmic examination (slit-lamp biomicroscopy before and after corneal application of fluorescein stain on both eyes) were performed every other day throughout the 30-day observation period. A modified ocular surface and respiratory tract clinical scoring system23 was used to quantify examination findings and calculate a cumulative clinical score, which could range from 0 to 17, on each examination day. Briefly, clinical signs of blepharospasm, ocular discharge, conjunctival hyperemia, and chemosis were each scored on a scale of 0 to 3, where 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Corneal epithelial ulceration was also scored on a scale of 0 to 3, where 0 = none, 1 = punctate ulcerations, 2 = 1 or more linear or dendritic ulcerations, and 3 = geographic ulcerations. When a cat had multiple classifications of corneal ulcerations present during an examination, it was assigned the score consistent with the most severe classification present. Nasal discharge and sneezing were scored as either 0 (absent) or 1 (present). The same investigator (ECL) performed all clinical examinations and clinical ocular disease score calculations. The respiratory rate of each cat was recorded every other day throughout the observation period beginning on day 0. The respiratory rate was the number of thoracic excursions during 1 minute and was calculated by visual observation of the cat through the cage door before the ophthalmic examination was performed (ie, while the cat was undisturbed). Blood (5 mL) for a CBC and serum biochemical analysis was collected by peripheral venipuncture from each cat on days 0, 15, and 30.
FHV-1 virus isolation
For each cat, conjunctival swab specimens for detection of FHV-1 by virus isolation and real-time PCR assay were collected from each eye at 3-day intervals beginning on day 0. The specimens were obtained after clinical ocular and respiratory disease scoring, but before fluorescein stain application. A sterile polyester-tipped swabi was gently brushed across the superior and inferior conjunctival fornices of each eye. Immediately after specimen collection, the swabs were placed in a sterile tube that contained virus transport medium composed of 10% neonatal calf serum and a 3× antibiotic-antimycotic solution in PBS solution. The tubes were incubated on ice for 4 hours and then vortexed for 15 seconds. For each tube, the FHV-1 titer in the transport medium was determined as described.20 Briefly, 6 sequential 10-fold dilutions of sample transport medium (inoculum) were added to monolayers of Crandell-Rees feline kidney cells in a 12-well plate and incubated for 2 hours. The inoculum medium was removed and replaced with minimal essential medium containing 0.88% carboxylmethylcellulose. The plates were incubated for 48 hours and then fixed, and the number of FHV-1 plaque-forming units/mL was determined for each well.
Relative FHV-1 genome quantification by real-time PCR assay
A real-time PCR assay was used to assess the genome of the FHV-1 isolates recovered from conjunctival swab specimen transport medium samples. Briefly, DNA was isolated from 200 μL of each transport medium sample by use of a commercial DNA extraction kitj in accordance with the manufacturer's recommended protocol and diluted to a concentration of 15 ng/μL. Feline herpesvirus-1 DNA was quantified by use of a previously described25 plasmid standard, which contained primers that targeted the US7 gene of the virus. To determine the number of feline genome equivalents in each transport medium sample, the fAlb (NM_001009961.2)26 was amplified with polymerasek (forward primer, 5′–TTCTTTAGCTGTCCGTGGTC-3′; reverse primer, 5′–CAAACTTCTGGGAGGTGATG-3′) and cloned into the pJET1.2 vector.l This plasmid was linearized with the restriction enzyme XbaI,l and a series of 10-fold dilutions ranging from 101 to 107 were prepared. The amplification efficiency of that standard was confirmed to be > 99% with R2 = 1.000 (forward primer, 5′–TTCTGCTCTGCAAGTCGATG-3′; reverse primer, 5′–TCTCAGCCTCAGGAAGTGTG-3′). The real-time PCR assay was performed by use of SYBR green reagentsm and a commercial PCR system, per the manufacturer's protocol.n Standard curves were generated and used to interpolate the viral and host genome equivalents. Results were expressed as the number of FHV-1 genome copies/10 copies of fAlb.
Confocal microscopy
In vivo confocal microscopic examinationso of the cornea were performed by use of a 63× objectivep and 400-μm field lens. Confocal microscopic examinations were performed on both eyes of each cat on days 0 (before FHV-1 inoculation) and 10. For each examination, a single drop of topical anestheticq was applied to each eye. Several drops of contact gelr were applied to the front of the microscope lens and ocular surface. A sterile, single-use polymethyl methacrylate caps mounted on the microscope lens was positioned perpendicular to and in slight contact with the ocular surface to obtain digital images. The polymethyl methacrylate cap was changed between cats.
Following completion of the examination, the digital confocal microscopic images were evaluated for evidence of pathological lesions. For each eye of each cat, 3 images of the basal corneal epithelium at the axial aspect of the cornea were assessed for signs of leukocyte infiltration. Within each image, the number of leukocytes per square millimeters of corneal tissue was quantified by use of semiautomated cell-counting software.t The mean leukocyte count for each eye was calculated and used for analysis purposes. All images were evaluated and all leukocyte counts were performed by the same investigator (ECL), who was unaware of the treatment assigned to each cat.
Statistical analysis
Data distributions for continuous outcomes of interest (clinical ocular disease score, respiratory disease score, cumulative clinical score, respiratory rate, FHV-1 titer, and relative FHV-1 genome quantification [No. of FHV-1 genome copies/10 copies of fAlb]) were assessed for normality by visual examination of frequency plot distributions. Linear least squares regression was used to assess the effect of treatment group (raltegravir or vehicle) and time on each continuous outcome of interest. Each model also included a random effect to account for repeated measures within cats.
The Kaplan-Meier method was used to compare duration of the FHV-1 shedding (as determined from virus isolation results) between the 2 treatment groups. A 2-sample Student t test was used to compare the mean corneal epithelial leukocyte count (No. of leukocytes/mm2 corneal tissue) between the 2 treatment groups for the left and right eyes on day 10. Fisher exact tests were used to compare the number of cats with an inflammatory leukogram (ie, neutrophilia with a left shift and lymphopenia) between the 2 groups on days 15 and 30 and to compare the number of cats that vomited after capsule administration between the 2 groups. All analyses were performed with statistical software,u and values of P < 0.05 were considered significant.
Results
Cats
None of the cats had remarkable physical and ophthalmologic examination findings or CBC and serum biochemical abnormalities on day 0. Five cats in the raltegravir group and 3 cats in the vehicle group had an inflammatory leukogram on day 15, whereas 2 cats in the raltegravir group and 1 cat in the vehicle group had an inflammatory leukogram on day 30. The number of cats with an inflammatory leukogram did not differ significantly between the 2 groups on day 15 (P = 0.30) or day 30 (P = 0.50). None of the cats had remarkable serum biochemical abnormalities on either day 15 or 30.
Four cats were observed to vomit several minutes after capsule administration on 5 days. Only 1 cat from each group vomited on any given day. One cat in the vehicle group vomited after capsule administration on day 4. One cat in the raltegravir group vomited after capsule administration on days 4, 6, and 9. Two other cats in the raltegravir group vomited after capsule administration once, and both of those occurrences happened early in the treatment period (before day 9). The number of cats that vomited after capsule administration did not differ significantly (P = 0.19) between the 2 treatment groups.
Clinical ocular and respiratory disease scores
Clinical signs consistent with ocular FHV-1 infection were detected in all cats in both treatment groups by day 4. Ocular disease was characterized by intermittent blepharospasm, mucopurulent ocular discharge, conjunctival hyperemia, chemosis, and corneal epithelial ulceration. Corneal ulceration was characterized by the presence of punctate, dendritic, and geographic superficial ulcers, and several cats had multiple morphological types of superficial corneal ulceration present on some days. Corneal ulcers were first detected in 4 cats in the raltegravir group and 3 cats in the vehicle group on day 6 and were present in all cats of the vehicle group on day 10 and all cats in the raltegravir group on day 12. For both groups, the number of cats with corneal ulceration began to slowly decline after day 16. Corneal ulcers were not detected in any of the cats on day 30 (end of the observation period).
Three cats in the raltegravir group and 2 cats in the vehicle group developed signs of upper respiratory tract disease, which was characterized by sneezing and the presence of nasal discharge, on day 6. None of the cats in the raltegravir group had signs of upper respiratory tract disease after day 16, except for 1 that had a nasal discharge on day 26. For cats in the vehicle group, signs of upper respiratory tract disease were not observed after day 26.
The cats in the raltegravir group developed moderate to severe conjunctivitis and corneal epithelial ulceration. The mean ± SD cumulative clinical score (ie, clinical ocular disease and respiratory disease scores combined; 10.7 ± 3.3) and clinical ocular score (9.7 ± 2.9) for the raltegravir group both peaked on day 8. The cats in the vehicle group developed moderate to severe conjunctivitis and corneal epithelial ulceration. The mean ± SD cumulative clinical score for the vehicle group (13.1 ± 2.8) peaked on day 10, whereas the mean ± SD clinical ocular score (12.0 ± 1.6) peaked on day 8. The mean cumulative clinical score for the raltegravir group was significantly (P = 0.023) less than that for the vehicle group, with the mean daily cumulative score differing by 1.36 between the 2 groups. The mean cumulative clinical score for the raltegravir group was consistently less than that for the vehicle group between days 8 and 26 (Figure 1). The clinical ocular disease scores mirrored the cumulative clinical disease scores.
Mean ± SD cumulative clinical score (A), respiratory rate (B), conjunctival FHV-1 titer (C), and relative FHV-1 genome quantification (No. of FHV-1 genome copies/10 copies of fAlb; D) over time for 6-month-old cats with experimentally induced ocular FHV-1 infection that were orally administered raltegravir (80 mg; dashed gray line; n = 7) or a vehicle (250 mg of lactose powder; solid black line; 7) every 12 hours for 14 days beginning on day 1. For each cat on day 0, 0.1 mL of a solution containing 106 plaque-forming units (PFUs) of FHV-1 strain FH2CS was topically applied to the inferior conjunctival fornix of each eye to experimentally induce an ocular FHV-1 infection. Each cat was observed for signs of ocular and respiratory tract disease every other day throughout the 30-day observation period beginning on day 0 (prior to experimental induction of ocular FHV-1 infection). Each cat was assigned a cumulative clinical score, which could range from 0 to 17, on each examination day. Briefly, clinical signs of blepharospasm, ocular discharge, conjunctival hyperemia, and chemosis were each scored on a scale of 0 to 3, where 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Corneal epithelial ulceration was also scored on a scale of 0 to 3, where 0 = none, 1 = punctate ulcerations, 2 = 1 or more linear or dendritic ulcerations, and 3 = geographic ulcerations. When a cat had multiple classifications of corneal ulcerations present during an examination, it was assigned the score consistent with the most severe classification present. Nasal discharge and sneezing were scored as either 0 (absent) or 1 (present). The respiratory rate of each cat was recorded by visual observation of thoracic excursions before the ophthalmic examination (ie, while the cat was undisturbed) every other day throughout the observation period beginning on day 0. Conjunctival swab specimens for determination of FHV-1 titer and relative FHV-1 genome quantification were obtained from each eye of each cat at 3-day intervals throughout the observation period beginning on day 0.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Mean ± SD cumulative clinical score (A), respiratory rate (B), conjunctival FHV-1 titer (C), and relative FHV-1 genome quantification (No. of FHV-1 genome copies/10 copies of fAlb; D) over time for 6-month-old cats with experimentally induced ocular FHV-1 infection that were orally administered raltegravir (80 mg; dashed gray line; n = 7) or a vehicle (250 mg of lactose powder; solid black line; 7) every 12 hours for 14 days beginning on day 1. For each cat on day 0, 0.1 mL of a solution containing 106 plaque-forming units (PFUs) of FHV-1 strain FH2CS was topically applied to the inferior conjunctival fornix of each eye to experimentally induce an ocular FHV-1 infection. Each cat was observed for signs of ocular and respiratory tract disease every other day throughout the 30-day observation period beginning on day 0 (prior to experimental induction of ocular FHV-1 infection). Each cat was assigned a cumulative clinical score, which could range from 0 to 17, on each examination day. Briefly, clinical signs of blepharospasm, ocular discharge, conjunctival hyperemia, and chemosis were each scored on a scale of 0 to 3, where 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Corneal epithelial ulceration was also scored on a scale of 0 to 3, where 0 = none, 1 = punctate ulcerations, 2 = 1 or more linear or dendritic ulcerations, and 3 = geographic ulcerations. When a cat had multiple classifications of corneal ulcerations present during an examination, it was assigned the score consistent with the most severe classification present. Nasal discharge and sneezing were scored as either 0 (absent) or 1 (present). The respiratory rate of each cat was recorded by visual observation of thoracic excursions before the ophthalmic examination (ie, while the cat was undisturbed) every other day throughout the observation period beginning on day 0. Conjunctival swab specimens for determination of FHV-1 titer and relative FHV-1 genome quantification were obtained from each eye of each cat at 3-day intervals throughout the observation period beginning on day 0.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Mean ± SD cumulative clinical score (A), respiratory rate (B), conjunctival FHV-1 titer (C), and relative FHV-1 genome quantification (No. of FHV-1 genome copies/10 copies of fAlb; D) over time for 6-month-old cats with experimentally induced ocular FHV-1 infection that were orally administered raltegravir (80 mg; dashed gray line; n = 7) or a vehicle (250 mg of lactose powder; solid black line; 7) every 12 hours for 14 days beginning on day 1. For each cat on day 0, 0.1 mL of a solution containing 106 plaque-forming units (PFUs) of FHV-1 strain FH2CS was topically applied to the inferior conjunctival fornix of each eye to experimentally induce an ocular FHV-1 infection. Each cat was observed for signs of ocular and respiratory tract disease every other day throughout the 30-day observation period beginning on day 0 (prior to experimental induction of ocular FHV-1 infection). Each cat was assigned a cumulative clinical score, which could range from 0 to 17, on each examination day. Briefly, clinical signs of blepharospasm, ocular discharge, conjunctival hyperemia, and chemosis were each scored on a scale of 0 to 3, where 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Corneal epithelial ulceration was also scored on a scale of 0 to 3, where 0 = none, 1 = punctate ulcerations, 2 = 1 or more linear or dendritic ulcerations, and 3 = geographic ulcerations. When a cat had multiple classifications of corneal ulcerations present during an examination, it was assigned the score consistent with the most severe classification present. Nasal discharge and sneezing were scored as either 0 (absent) or 1 (present). The respiratory rate of each cat was recorded by visual observation of thoracic excursions before the ophthalmic examination (ie, while the cat was undisturbed) every other day throughout the observation period beginning on day 0. Conjunctival swab specimens for determination of FHV-1 titer and relative FHV-1 genome quantification were obtained from each eye of each cat at 3-day intervals throughout the observation period beginning on day 0.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
The mean respiratory rate for the raltegravir group was also significantly (P = 0.034) lower than that for the vehicle group over time (Figure 1). None of the cats required rescue analgesia during the observation period.
FHV-1 isolation
Feline herpesvirus-1 was isolated from conjunctival swab specimens obtained from all cats on day 3. For the cats in the raltegravir group, FHV-1 was isolated from all 7 cats through day 15, after which the proportion of cats that were shedding the virus in that group decreased fairly rapidly, with only 1 cat continuing to shed FHV-1 between days 24 and 30. For the cats in the vehicle group, FHV-1 was isolated from all 7 cats through day 21, 5 cats on day 24, 2 cats on day 27, and 1 cat on day 30. The mean conjunctival FHV-1 titer did not differ significantly (P = 0.26) between the 2 treatment groups (Figure 1). However, the median duration of FHV-1 shedding for the raltegravir group (21 days) was significantly (P = 0.05) shorter than that for the vehicle group (27 days; Figure 2).
Kaplan-Meier plot that depicts the percentage of cats from Figure 1 that were shedding FHV-1 over time throughout the 30-day observation period. Feline herpesvirus-1 shedding was determined on the basis of virus isolation results. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Kaplan-Meier plot that depicts the percentage of cats from Figure 1 that were shedding FHV-1 over time throughout the 30-day observation period. Feline herpesvirus-1 shedding was determined on the basis of virus isolation results. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Kaplan-Meier plot that depicts the percentage of cats from Figure 1 that were shedding FHV-1 over time throughout the 30-day observation period. Feline herpesvirus-1 shedding was determined on the basis of virus isolation results. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Relative FHV-1 genome quantification by real-time PCR assay
All study cats had positive real-time PCR assay results (ie, FHV-1 DNA was detected in conjunctival swab specimens) on day 3. For cats in the raltegravir group, FHV-1 DNA was detected in conjunctival swab specimens from all 7 cats through day 21, from 6 cats on day 24, from 3 cats on day 27, and from all 7 cats on day 30. For cats in the vehicle group, FHV-1 DNA was detected in conjunctival swab specimens from all 7 cats through day 24, from 5 cats on day 27, and from all 7 cats on day 30 (Figure 1). The mean number of FHV-1 genomes/10 copies of fAlb did not differ significantly (P = 0.85) between the 2 groups at any time throughout the observation period.
Confocal microscopy
In vivo confocal microscopy revealed that leukocyte infiltration of the corneal epithelium (keratitis) was variable in all cats on day 10 (Figure 3). The mean ± SD corneal epithelial leukocyte count was 120 ± 106 leukocytes/mm2 and 128 ± 98 leukocytes/mm2 for the right and left eyes, respectively, of cats in the raltegravir group and 155 ± 114 leukocytes/mm2 and 119 ± 116 leukocytes/mm2 for the right and left eyes, respectively, of cats in the vehicle group. The mean corneal epithelium leukocyte count did not differ significantly (P = 0.32) between the 2 groups at any time.
Representative in vivo confocal photomicrographs of the corneal epithelium on day 10 for 2 cats described in Figure 1 that received either the raltegravir (A) or vehicle (B) treatment. Notice that leukocytes (arrows) have infiltrated the corneal epithelium of each cat. Bar = 50 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Representative in vivo confocal photomicrographs of the corneal epithelium on day 10 for 2 cats described in Figure 1 that received either the raltegravir (A) or vehicle (B) treatment. Notice that leukocytes (arrows) have infiltrated the corneal epithelium of each cat. Bar = 50 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Representative in vivo confocal photomicrographs of the corneal epithelium on day 10 for 2 cats described in Figure 1 that received either the raltegravir (A) or vehicle (B) treatment. Notice that leukocytes (arrows) have infiltrated the corneal epithelium of each cat. Bar = 50 μm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.490
Discussion
In the present study, the duration of conjunctival FHV-1 shedding (as determined by virus isolation) was shorter and signs of clinical ocular and respiratory tract disease were less severe in cats that received raltegravir (80 mg, PO, q 12 h for 14 days), compared with cats that received a lactose powder placebo (vehicle). However, virus isolation and real-time PCR assay results suggested that the FHV-1 titer did not differ significantly between raltegravir- and vehicle-treated cats with experimentally induced ocular FHV-1 infections. Additionally, the corneal epithelial leukocyte count did not differ significantly between the raltegravir- and vehicle-treated cats. Cats in both treatment groups developed moderate to severe conjunctivitis and keratitis, and the severity of clinical ocular disease may have impaired our ability to detect subtle differences in the extent of corneal epithelial leukocyte infiltration between the 2 groups.
It is important to note that, although the mean clinical ocular disease score for the raltegravir group of this study was significantly lower than that for the vehicle group, all cats in both groups developed fairly severe ocular disease. In the present study, the dose of raltegravir administered on a per-kilogram-of-body-weight basis (mean ± SD, 27.3 ± 3.4 mg/kg; range, 23 to 32 mg/kg) was similar to but slightly higher than that administered to FeLV-infected cats of another study19 (20 to 25 mg/kg). In that study,19 as in the present study, the raltegravir capsules were prepared prior to study initiation, and normal biological variation in the body weight of study subjects resulted in variation in the dose of raltegravir administered throughout the treatment period. Ideally, each cat would have been administered the same dose of raltegravir (on a mg/kg basis) throughout the treatment period, but that could not be practically achieved by oral administration of the drug in capsules. Nevertheless, variation in the raltegravir dose administered to study subjects throughout the treatment period accurately reflects clinical practice where patients are routinely administered oral medications at doses within a therapeutic range to accommodate available drug formulations and normal variation in patient body weight. Further research is necessary to determine the optimal raltegravir dosing schedule for cats with ocular and respiratory FHV-1 infections. In particular, the pharmacokinetics and pharmacodynamics of raltegravir in cats when various formulations of the drug are administered at various doses and by different routes need to be elucidated.
In human subjects, the half-life of raltegravir ranges from 7 to 12 hours,16 and glucuronidation is the primary mechanism by which raltegravir is cleared from the body.27 The glucuronidation capacity of cats is low; therefore, cats may be more susceptible than humans to raltegravir toxicosis owing to decreased clearance and tissue accumulation of the drug.17,27 In cats with progressive FeLV infection, raltegravir was well tolerated at dosages as high as 80 mg/cat, PO, every 12 hours for 2.5 weeks.19 The raltegravir-treated cats of the present study received 80 mg of the drug, PO, every 12 hours for 2 weeks, and no overt signs of systemic toxicosis were observed. However, effects associated with long-term (> 2 weeks) administration of raltegravir to cats warrant further investigation.
Globally, there is little genomic variation among FHV-1 strains, with only 3 main genotypes recognized.1,5,28–30 However, there is substantial variation in virulence among field isolates of the same strain of FHV-1,1,8 and that variation in virulence may explain, at least in part, the severity of the clinical signs observed in the cats of the present study (ie, the FHV-1 strain used in this study appeared to be quite virulent). Drug-resistant strains and mutations of herpesviruses have been identified and studied to help elucidate viral enzyme structure and function, identify potential antiviral mechanisms, and understand clinical drug resistance.31,32 In vitro exposure of herpesviruses to increasing concentrations of antiviral agents known to be ineffective at inhibiting viral replication has resulted in drug-resistant strains of the viruses.8,31 Thus, in vivo studies are critical for the development of alternate antiviral therapies and establishment of effective antiviral dosing protocols to inhibit viral replication and decrease the frequency of virus mutation and development of chemical resistance. Clinical evaluation of integrase inhibitor treatment failures in human patients is limited, but in vitro data suggest that resistance to raltegravir is usually the result of multiple integrase gene substitutions.16
The cats of the present study were healthy immediately prior to study initiation and experimental inoculation of FHV-1; therefore, this research model may not have accurately mimicked all clinical scenarios by which FHV-1 infection can manifest in cats. Another limitation of the present study was that the number of cats evaluated in each treatment group was fairly small (n = 7). Also, ocular shedding of FHV-1 might have been missed or the viral load underestimated in some cats because conjunctival swab specimens for detection of FHV-1 by virus isolation and real-time PCR assay were obtained only at 3-day intervals throughout the observation period. In a study33 of 25 HSV-2 seropositive and 18 HSV-1 seropositive immunocompetent adult human patients, quantitative PCR assay results of serially collected swab specimens indicated that 24% of anogenital HSV-2 reactivations and 21% of oral HSV-1 reactivations lasted ≤ 6 hours and 49% of anogenital HSV-2 reactivations and 39% of oral HSV-1 reactivations lasted ≤ 12 hours. It is possible that more frequent collection of conjunctival swab specimens than at 3-day intervals may have allowed us to detect FHV-1 more frequently or at higher titers than those reported.
Results of the present study suggested that raltegravir, an integrase inhibitor, might represent a novel antiviral agent for the treatment of ocular and respiratory tract FHV-1 infections in cats. Further research is necessary to determine the efficacy of raltegravir for the treatment of clinical patients and shelter cats with naturally acquired FHV-1 infections as well as to identify the optimal raltegravir dosing protocol (eg, drug formulation, dose, and route and frequency of administration) for use in such cats.
Acknowledgments
Supported by the Foundation for Ophthalmology Research and Education International.
ABBREVIATIONS
| fAlb | Feline albumin gene |
| FHV-1 | Feline herpesvirus-1 |
| HSV | Herpes simplex virus |
Footnotes
Companion Animal Hospital, College of Veterinary Medicine, Cornell University, Ithaca, NY.
Isentress, 400-mg film-coated tablets, Merck and Co, Kenilworth, NJ.
Lactose powder, PCCA, Houston, Tex.
Schirmer tear test standardized sterile strips, Intervet Inc, Summit, NJ.
Kowa SL-15, Kowa Co, Tokyo, Japan.
Ful-Glo Fluorescein Sodium Strips, USP, Moore Medical LLC, Farmington, Conn.
Tono-Pen XL, Reichert Inc, Depew, NY.
Omega 500, Heine Optotechnik, Herrsching, Germany.
Sterile polyester-tipped applicators, Puritan Medical Products Co, Guilford, Me.
DNeasy Blood and Tissue, Qiagen, Valencia, Calif.
iProof polymerase, Bio-Rad Laboratories Inc, Hercules, Calif.
Thermo Fisher Scientific Inc, Waltham, Mass.
SYBR green reagents, Thermo Fisher Scientific Inc, Waltham, Mass.
ABI 7500 Fast Real-Time PCR System, Thermo Fisher Scientific Inc, Waltham, Mass.
Heidelberg Engineering, Heildelberg, Germany.
Carl Zeiss Meditec AG, Jena, Germany.
Proparacaine hydrochloride 0.5% ophthalmic solution, USP, Akorn, Lake Forest, Ill.
GenTeal tear gel, Novartis Pharmaceuticals Corp, East Hanover, NJ.
TomoCap, Heidelberg Engineering, Heidelberg, Germany.
Rostock Cornea Module Software, version 1.3.3, Heidelberg Engineering, Heidelberg, Germany.
SPSS, version 24, IBM Corp, White Plains, NY.
References
1. Gould D. Feline herpesvirus-1: ocular manifestations, diagnosis and treatment options. J Feline Med Surg 2011;13:333–346.
2. Maggs DJ, Lappin MR, Reif JS, et al. Evaluation of serologic and viral detection methods for diagnosing feline herpesvirus-1 infection in cats with acute respiratory tract or chronic ocular disease. J Am Vet Med Assoc 1999;214:502–507.
3. Andrew SE. Ocular manifestations of feline herpesvirus. J Feline Med Surg 2001;3:9–16.
4. Gaskell R, Dawson S, Radford A, et al. Feline herpesvirus. Vet Res 2007;38:337–354.
5. Gaskell RM, Povey RC. Experimental induction of feline viral rhinotracheitis virus re-excretion in FVR-recovered cats. Vet Rec 1977;100:128–133.
6. Stiles J. Ocular manifestations of feline viral diseases. Vet J 2014;201:166–173.
7. Thomasy SM, Lim CC, Reilly CM, et al. Evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type-1. Am J Vet Res 2011;72:85–95.
8. 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):119–130.
9. Thomasy SM, Shull O, Outerbridge CA, et al. Oral administration of famciclovir for treatment of spontaneous ocular, respiratory, or dermatologic disease attributed to feline herpesvirus type 1: 59 cases (2006–2013). J Am Vet Med Assoc 2016;249:526–538.
10. Fontenelle JP, Powell CC, Veir JK, et al. Effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats. Am J Vet Res 2008;69:289–293.
11. Yan Z, Bryant KF, Gregory SM, et al. HIV integrase inhibitors block replication of alpha-, beta-, and gammaherpesviruses. MBio 2014;5:e01318–e14.
12. Karade SK, Ghate MV, Chaturbhuj DN, et al. Cross-sectional study of virological failure and multinucleoside reverse transcriptase inhibitor resistance at 12 months of antiretroviral therapy in Western India. Medicine (Baltimore) 2016;95:e4886.
13. Nadal M, Mas PJ, Blanco AG, et al. Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain. Proc Natl Acad Sci U S A 2010;107:16078–16083.
14. Zhou B, Yang K, Wills E, et al. A mutation in the DNA polymerase accessory factor of herpes simplex virus 1 restores viral DNA replication in the presence of raltegravir. J Virol 2014;88:11121–11129.
15. Zhukovskaya NL, Guan H, Saw YL, et al. The processivity factor complex of feline herpes virus-1 is a new drug target. Antiviral Res 2015;115:17–20.
16. Hicks C, Gulick RM. Raltegravir: the first HIV type 1 integrase inhibitor. Clin Infect Dis 2009;48:931–939.
17. Cattori V, Weibel B, Lutz H. Inhibition of feline leukemia virus replication by the integrase inhibitor raltegravir. Vet Microbiol 2011;152:165–168.
18. Greggs WM III, Clouser CL, Patterson SE, et al. Discovery of drugs that possess activity against feline leukemia virus. J Gen Virol 2012;93:900–905.
19. Boesch A, Cattori V, Riond B, et al. Evaluation of the effect of short-term treatment with the integrase inhibitor raltegravir (Isentress) on the course of progressive feline leukemia virus infection. Vet Microbiol 2015;175:167–178.
20. Pennington MR, Fort MW, Ledbetter EC, et al. A novel corneal explant model system to evaluate antiviral drugs against feline herpesvirus type 1 (FHV-1). J Gen Virol 2016;97:1414–1425.
21. 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-vision-research/. Accessed Oct 23, 2018.
22. Ledbetter EC, Dubovi EJ, Kim SG, et al. Experimental primary ocular canine herpesvirus-1 infection in adult dogs. Am J Vet Res 2009;70:513–521.
23. 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;31:642–649.
24. Walton TE, Gillespie JH. Feline viruses. VII. Immunity to the feline herpesvirus in kittens inoculated experimentally by the aerosol method. Cornell Vet 1970;60:232–239.
25. Pennington MR, Van de Walle GR. Electric cell-substrate impedance sensing to monitor viral growth and study cellular responses to infection with alphaherpesviruses in real time. mSphere 2017;2:e00039–17.
26. Helfer-Hungerbuehler AK, Widmer S, Hofmann-Lehmann R. GAPDH pseudogenes and the quantification of feline genomic DNA equivalents. Mol Biol Int 2013;2013:587680.
27. Kassahun K, McIntosh I, Cui D, et al. Metabolism and disposition in humans of raltegravir (MK-0518), an anti-AIDS drug targeting the human immunodeficiency virus 1 integrase enzyme. Drug Metab Dispos 2007;35:1657–1663.
28. Kolb AW, Lewin AC, Moeller Trane R, et al. Phylogenetic and recombination analysis of the herpesvirus genus varicellovirus. BMC Genomics 2017;18:887.
29. Vaz PK, Job N, Horsington J, et al. Low genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1. BMC Genomics 2016;17:704.
30. Lewin AC, Kolb AW, McLellan GJ, et al. Genomic, recombinational and phylogenetic characterization of global feline herpesvirus 1 isolates. Virology 2018;518:385–397.
31. Sarisky RT, Quail MR, Clark PE, et al. Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir. J Virol 2001;75:1761–1769.
32. Coen DM. The implications of resistance to antiviral agents for herpesvirus drug targets and drug therapy. Antiviral Res 1991;15:287–300.
33. Mark KE, Wald A, Magaret AS, et al. Rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults. J Infect Dis 2008;198:1141–1149.
