Feline herpesvirus-1 is distributed worldwide in the cat population. The prevalence in cats in a colony is higher (> 70%) than the prevalence in cats in singlecat households (< 50%).1 This alphaherpesvirus is recognized as one of the most important pathogens causing respiratory tract infections, conjunctivitis, and keratitis in cats.2–4 Feline herpesvirus-1 typically infects and replicates in epithelial tissues of the respiratory tract and conjunctiva, causing cytopathic lesions. It has been reported that about 80% of cats are unable to eliminate the virus after primary infection and will develop a carrier state.5 During latency, virus frequently persists in the trigeminal ganglion and possibly in the cornea.6–8 The disease can be reactivated following stress or corticosteroid treatment.8–10 It remains difficult to successfully treat cats with chronic ocular manifestations of FHV-1 infection, such as corneal dendritic ulcers and intermittent conjunctivitis.
The IFNs are members of a cytokine family that play an important role as mediators of innate (nonspecific) immunity. Among their multifunctional properties, they have antiviral, antiproliferative, and immune regulatory functions. The IFNs can be classified on the basis of biological and chemical properties into 4 antigenically distinct groups (ie, IFN-α, IFN-β, IFN-γ, and IFN-ω). In vitro and in vivo studies in humans, monkeys, and rabbits have revealed antiviral effects of IFN-α against keratitis caused by herpes simplex virus 1 when used alone11–15 or in combination with nucleoside analogues, such as trifluridine,16 idoxuridine, or acyclovir.17–19 The antiviral effect was greatest when IFN was administered at high concentrations (1 X 106 to 15 X 106 U/mL).12–15,20 Topical application of IFN-á before or shortly after infection with herpes simplex virus 1 reduces the severity and duration of clinical signs19 and significantly shortens the period of virus shedding.20
Although only minimal data are available to support the use of rHuIFN-α in the treatment of cats,21,a this drug has been recommended for the treatment of cats with FHV infection.22,a A once-daily dose orally administered to cats results in less severe clinical signs, compared with severity of signs for an untreated control group.a Other information on the benefits of a topical, low-dose IFN treatment for cats with FHV-1–induced ocular lesions is anecdotal and based on unpublished observations.
An rFeIFN-ω product is currently available commercially,23 and a few initial clinical trials have been conducted24,25 to assess the antiviral properties of rFeIFN-ω in dogs and cats with viral infections. In 1 study,b investigators tested the in vitro antiviral effects of rFeIFN-ω at a concentration of 50,000 U/mL on FHV-1–infected cells and reported a significant reduction of virus titers. In another study,26 investigators examined the inhibitory effect of a low dose of rFeIFN-ω on in vitro replication of feline enteropathogenic viruses and observed that IFN inhibited the replication of some of the viruses tested. In that study, the investigators used rFeIFN-ω concentrations of 100 and 1,000 U/mL.
The study reported here was conducted to evaluate the antiviral efficacy of a wide range of concentrations (100 to 500,000 U/mL) of rFeIFN-ω and rHuIFN-α2b on in vitro replication of FHV-1 in cultures of CRFK cells. This range of concentrations was chosen to validate the effects of concentrations used in other studies, to determine whether the antiviral activity was a dose-dependent event, and to compare the optimal antiviral concentrations of rFeIFN-ω and rHuIFN-α2b against FHV-1. Furthermore, a novel method for assessing antiviral efficacy of IFN on FHV-1 by measurement of reduction in plaque size was used in this study. In vitro conversion of MTT dye was used to exclude the possibility of direct cytotoxic effects of IFN preparations on cell cultures that could confound analysis of antiviral effects. The species-specificity of type I IFNs has been reported,21 and maximal antiviral activity is expected in homologous cells. Therefore, we hypothesized that rFeIFN-ω would have a more profound effect than would rHuIFN-α2b on FHV-1 replication in CRFK cells.
Materials and Methods
Sample population—The CRFK cellsc were purchased from the ATCC. Cells were maintained in culture conditions recommended by the ATCC. For all experiments, cells were used between passages 9 through 13.
Virus—Feline herpesvirus-1d was obtained from the ATCC. The virus dilution (10−6, which yielded an approx titer of 102.7 TCID50/mL) resulted in a mean of 320 PFU/mL for the experiments. This virus concentration was chosen as the experimental challenge dose because plaques were distinct and plaque counts had minimal variability (SEM, ± 11.9 PFU/mL).
IFN products—The rHuIFN-α2be and rFeIFN-ωf were obtained as a lyophilized preparation. Lyophilized powders were dissolved in isotonic saline (0.9% NaCl) solution, and the prepared IFN solutions were stored at 2° to 4°C.
Assessment of the number and size of plaques— Confluent monolayers of CRFK cells were grown in 24-well culture plates during an incubation period of 24 hours. Cells were treated with rFeIFN-ω or rHuIFN-α2b at concentrations of 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 100,000, 250,000, and 500,000 U/mL. Because of the inclusion of washes with PBS solution and use of the overlay technique, IFN was added at 3 time points to maintain constant concentrations throughout the study. Therefore, cells were treated with IFN 6 hours before the addition of FHV-1, concurrent with the addition of FHV-1 and concurrent with addition of the overlay medium. One milliliter of a solution containing FHV-1 (32 PFU/mL) was added to each well. A solution of 2X Dulbecco modified Eagle medium (diluted 1:1 with 1.6% agarose) was used as the overlay medium. All treatment and control wells were performed in duplicate. Control cell cultures were treated exactly the same as test cell cultures, except that PBS solution was added instead of IFN solution.
After incubation for 72 hours, cells were fixed by use of a 10% formalin solution. The overlay medium was removed, and remaining cells were stained with crystal violet. Plaques were counted by use of an inverted ocular microscope, and the mean number of plaques was calculated from 2 wells/dilution for each assay. All assays were repeated 6 times to establish reproducibility.
For the evaluation of plaque size, plaques in 20 cultures treated with 100,000 U/mL, 40 cultures treated with 250,000 U/mL, 40 cultures treated with 500,000 U/mL, and 100 control cultures were examined microscopically. Diameter of each plaque was measured by use of a measuring reticle. Size of plaques for IFN-treated cultures was analyzed and compared with size of plaques for control cultures.
Cytotoxic assay—Events that adversely affect cell viability can be measured as a decrease in the reduction of MTT to formazan, and the colored product can be quantified by use of spectrophotometry. Two assays with 16 replicates/dilution were performed in 96-well culture plates of confluent CRFK cells. In each well, growth medium was supplemented with rFeIFN-ω or rHuIFN-α2b at the same dilutions that were used in the antiviral assays. Sets of 16 viable control wells received growth medium only, and 16 cell-death control wells were treated with 100% ethanol during the 12-hour incubation period. Plates were incubated at 37°C for 12 hours. Medium was aspirated and replaced with 100 μL of medium containing MTT solution (0.5 mg/mL). After incubation for 5 hours, 90 μL of the medium in each well was removed and replaced with 90 μL of solubilization solution (0.1N HCL-isopropanol); plates were then incubated for 5 minutes. A multiple-channel pipette was used to dissolve formazan crystals by mixing contents of each well. The mean corrected OD was calculated for each dilution and compared with values of both control groups.
Statistical analysis—Linear regression analysis was used to evaluate the optimal dosage for the virus challenge and to perform dose-response analysis of plaque counts. Doses were logarithmically transformed before linear regression analysis. A small value (0.01) was added to all concentrations to adjust for control values in the logarithmically transformed analysis. Linear regression analysis was performed by use of a commercially available software package.g Changes in plaque number and size and corrected OD values obtained for MTT assay means were compared by use of a Dunnett test. A 1-way ANOVA was performed by use of commercially available statistical software.h Significance for all analyses was set at values of P ≤ 0.05.
Results
Reductions in the number of plaques—Mean plaque number for the 12 concentrations of each IFN and the control cultures (untreated CRFK cells) were calculated (Figure 1). None of the treatments resulted in complete suppression of plaque development. The effect of the IFNs on plaque formation was assessed statistically for each IFN by use of the Dunnett procedure for testing multiple treatments against a control sample. The rFeIFN-ω treatments of 100,000 and 500,000 U/mL resulted in significant reductions in the number of plaques (54.7% and 59.8%, respectively). Linear regression analysis of the mean plaque number for IFN treatments, relative to the control values, revealed a significant correlation (R2 = 0.310; P < 0.001) for the entire dose range of rFeIFN-ω treatments (Figure 2). None of the concentrations of rHuIFN-α2b resulted in a significant reduction in the number of plaques.
Mean ± SEM number of plaques for CRFK cell cultures inoculated with FHV-1 and not treated with IFN (control [white bar]) or treated with various concentrations of rFeIFN-ω (black bars) or rHuIFN-α2b (gray bars). *Value differs significantly (P < 0.05), compared with the value for the control culture.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1406
Number of plaques in relation to the dose of rFeIFN-ω in culture medium of CRFK cells. Results reported for each dose of rFeIFN-ω are the mean ± SEM percentage of the number of plaques for the control culture. The control culture was assigned a value of 100%. Linear regression analysis revealed a significant correlation (R2 = 0.310; P < 0.001) for the effect of rFeIFN-ω at various concentrations. The line of best fit (solid line) and 95% confidence intervals (dotted lines) are indicated. *Within a concentration, the value differs significantly (P < 0.05), compared with the value for the control culture.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1406
Reductions in plaque size—Mean plaque size for each concentration of IFN, expressed as a percentage of the mean values for the control culture (infected but untreated CRFK cells), was calculated. Reductions in plaque size were induced by rFeIFN-ω and rHuIFN-α2b (Figure 3). Plaques were significantly smaller at all concentrations of rFeIFN-ω and rHuIFN-α2b, relative to plaque size for untreated control cultures. Mean reduction in plaque size for cells treated with rFeIFN-ω was 47.5% at 100,000 U/mL, 81.0% at 250,000 U/mL, and 70.5% at 500,000 U/mL. Mean reduction in plaque size for cells treated with rHuIFN-α2b was 56.4% at 100,000 U/mL, 75.7% at 250,000U/mL, and 69.8% at 500,000 U/mL.
Reduction in plaque size for CRFK cell cultures inoculated with FHV-1 and not treated with IFN (control [white bars]) or treated with various concentrations of rFeIFN-ω (black bars) or rHuIFN-α2b (gray bars). Results reported for each dose of IFN are expressed as the mean ± SEM percentage of the size for control plaques. *Within a concentration, the value differs significantly (P < 0.001), compared with the value for the control culture.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1406
To provide additional evidence of the difference in plaque size induced by IFN, photographs were taken of plaques and used for comparison (Figure 4). Plaques from cultures treated with rHuIFN-α2b and rFeIFN-ω were smaller, compared with the size of plaques for FHV-1–infected, untreated, CRFK cell cultures.
Photomicrographs of plaques (within white dotted lines) in cultures of CRFK cells inoculated with FHV-1 and not treated with IFN (A) or treated with the same concentration (500,000 U/mL) of rFeIFN-ω (B) or rHuIFN-α2b (C). Notice that uninhibited cell-to-cell spread results in plaques that are significantly larger, compared with the plaque sizes for cultures treated with IFN. Crystal violet stain; bar = 10 μm.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1406
Cytotoxic assay—Neither rFeIFN-ω nor rHuIFN-α2b treatment caused significant cellular toxicosis at the concentrations tested for viral inhibition (500, 5,000, 50,000, and 500,000 U/mL). The OD of cells treated with rFeIFN-ω or rHuIFN-α2b did not differ significantly from the OD of cells cultured in medium alone (viable control cells) but were all significantly greater than the OD for cells cultured in medium and treated with 100% methanol (cell-death control cells; Figure 5).
Results for an MTT assay to determine cytotoxic effects of rHuIFN-α2b and rFeIFN-ω on cultures of CRFK cells. Mean ± SEM OD values are reported for cultures of untreated cells (viable control cells [white bar]), cells treated with 100% methanol (cell-death control cells [striped bar]), cells treated with rFeIFN-ω (black bars), and cells treated with rHuIFN-α2b (gray bars). *Value differs significantly (P < 0.05), compared with the value for the cell-death control cells.
Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1406
Discussion
On the basis of analysis of the results reported here, the antiviral effect of rFeIFN-ω was greater than the effect induced by rHuIFN-α2b. This effect was found to be significantly greater only at some of the higher concentrations of IFNs; reductions in the number of plaques and size of plaques were observed. In addition and in agreement with 2 other reports,26,b the antiviral activity of rFeIFN-ω was found to be a dose dependent phenomenon. However, in contrast to those studies, the overall antiviral effect of rFeIFN-ω in the study reported here was considerably reduced.
Comparison of our results with results for the 2 aforementioned studies26,b is problematic because of differences in the type of assays and treatment protocols used. The choice of viruses and cells used for culture could have had a major influence on measurement of antiviral effects of the IFNs. The susceptibility of cell cultures to IFN varies considerably, depending on the cell line and preparation of IFN used.27,28 In other in vitro studies,26,b investigators have used fcwf-4 cells, which appear to be 10 times as sensitive to rFeIFN-ω, compared with CRFK cells. It has been speculated that fcwf-4 cells may possess more IFN receptors than CRFK cells; thus, fcwf-4 cells are more sensitive to inhibitory effects of IFN on cell growth.26 The number of cellular receptors for IFN can vary considerably among cell types (from 10/cell to 2,000/cell).29 Also, biological effects (such as initiation of cellular activation by receptor binding and differential viral binding affinity) may influence the mechanism of antiviral efficacy of IFN.
In the study reported here, treatment with rHuIFN-α2b did not result in a significant reduction in the number of plaques, compared with the number of plaques for control cultures. However, at higher concentrations of rHuIFN-α2b, a significant difference in antiviral activity was observed in the form of a reduction in plaque size. The reduction in plaque size was similar for rHuIFN-α2b and rFeIFN-ω. The greater antiviral activity of rFeIFN-ω, compared with the antiviral activity of rHuIFN-α2b, on the cell cultures was expected because species specificity of type I IFN is recognized and maximal antiviral activity typically results for homologous cells.21
It is possible that the plaque reduction assay used in our study was not sufficiently sensitive to detect the antiviral effect of rHuIFN-α2b or that homologous IFN is better at preventing viral replication than at influencing adjacent cells to resist viral infection. In other studies,24,25 investigators have used virus-yield reduction assays to test for the antiviral activity of rFeIFN-ω against FHV-1. It is currently unknown whether clinical results correlate best with the results obtained from yield reduction assays or with the plaque reduction assay used in the study reported here. Also, to avoid the potential for biased results, it would have been advantageous in our experiment to make the investigator who evaluated results of the assays (NS) unaware of the treatments for the cultures because the investigator who conducted the experiment and evaluated results of the assays was the same person.
None of the treatments with rHuIFN-α2b or rFeIFN-ω caused significant toxicosis of CRFK cells. Therefore, the antiviral activity for both IFNs could not be attributed to an in vitro effect on the viability or physiologic function of CRFK cells. Lack of cytotoxicosis in this in vitro cell system does not necessarily exclude in vivo toxicosis.
The 2 measures of antiviral activity (number of plaques and plaque size) used in the study should monitor different aspects of the activity of IFN. One action of IFN is to inhibit viral replication within infected cells. This effect is best monitored by a reduction in the number of plaques. A second mechanism of IFN is to protect uninfected cells from infection with a virus released adjacently. This action will affect the extent to which viral infection can spread from cell to cell, which directly affects plaque size. Treatment with rHuIFN-α2b or rFeIFN-ω had similar effects on reduction in plaque size. This suggests that each had a strong impact on the cell-to-cell spread of progeny virus, which indicated a similar ability to protect uninfected adjacent cells. On the other hand, the strong effect of rFeIFN-ω on the reduction of the number of plaques would indicate that this IFN has a greater ability to inhibit viral replication within infected CRFK cells.
The predictive value for a clinical application on the basis of in vitro studies is limited. In vivo, IFN must be effective under dynamic physiologic conditions within the body, and IFN may face substantial variability in metabolic conditions of ill animals. Nevertheless, analysis of the results of the in vitro study reported here suggests that rFeIFN-ω or rHuIFN-α2b may both be of therapeutic clinical value when administered at high doses early during FHV-1 infection. However, because the overall antiviral effect of both IFNs was very modest, IFNs may be most effective when administered as an adjunct treatment in combination with other antiviral agents, rather than being applied as a single therapeutic product.16–19 Use of rFeIFN-ω is likely to be more effective than an IFN of human origin.
The ability of the test compounds to inhibit cell-to-cell spread of virus may be relevant for clinical applications, such as topical ophthalmic treatment of cats with FHV-1–induced dendritic keratitis and conjunctivitis. It is likely that inhibition of viral spread will act to restrict the development of cytopathic lesions in corneal cells. This possibility needs to be tested directly because the study reported here was conducted in vitro and also because the cell culture used did not directly model the intended clinical application (ie, cornea cells). Testing that involves the use of feline corneal cells would be advantageous in determining the applicability of the IFN effects and could be instituted in future studies. It is postulated that the antiviral effect of IFN in corneal cells may be greater than was observed here. Corneal cells do not produce the large amounts of protease that are generated by renal cells.
Findings for the study reported here were supported by results of 2 other studiesi,j in which investigators evaluated the clinical antiviral activity of rFeIFN-ω in cats with FHV-1–induced ocular lesions. In one of those studies,i investigators detected the most profound clinical improvement in ocular FHV-1 manifestations and signs following topical treatment with rFeIFN-ω administered at a wide range of concentrations. The greatest effect was observed for the high dose of 500,000 U/mL. This confirms the results obtained in our study. In another study,j investigators examined the activity of topically and orally applied IFN-ω on the response of conjunctival and WBCs of cats as indicated by expression of Mx-protein. The effective dose was similar to that observed in the study reported here. Furthermore, prolonged biological activity was observed when rFeIFN-ω was administered at high doses. Antiviral activity was a dose-dependent event in both of those aforementioned studies.i,j By evaluating all available evidence, it may be concluded that rFeIFN-ω will be most effective when administered at high concentrations and that low doses of IFN may be of lesser value than has been suggested.3,a
Although the use of heterologous rHuIFN-α2b has been widely used for the treatment of cats with FHV-1–induced ocular lesions, the clinical value of this drug remains questionable, especially when it is used at low doses. Furthermore, there is the issue regarding whether long-term use of rHuIFN-α2b will continue to be of clinical benefit because the continued application of heterologous IFNs can induce the formation of cytokine-specific neutralizing antibodies.30
In the study reported here, we were able to detect anti–FHV-1 activity of IFNs by use of high concentrations of rFeIFN-ω or rHuIFN-α2b in vitro. Treatment with high doses of rFeIFN-ω or rHuIFN-α2b resulted in significant reductions in plaque size, which indicated potential efficacy for reducing the dimensions of FHV-1–induced cytopathic lesions. On the basis of analysis of our results, rFeIFN-ω may provide an effective adjunctive treatment for cats with FHV-1–induced lesions and may prove more beneficial than the use of heterologous rHuIFN-α2b. Treatment with rHuIFN-α2b or rFeIFN-ω did not cause significant cellular toxicosis in CRFK cells.
ABBREVIATIONS
| FHV | Feline herpesvirus |
| IFN | Interferon |
| rHuIFN-α | Recombinant human IFN-α |
| rFeIFN-ω | Recombinant feline IFN-ω |
| CRFK | Crandell-Rees feline kidney |
| MTT | 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide |
| ATCC | American Type Culture Collection |
| PFU | Plaque-forming units |
| OD | Optical density |
| fcwf | Felis catuswhole fetus |
Nasisse MP, Halenda RM, Luo H. Efficacy of low-dose oral, natural human interferon alpha in acute feline herpesvirus-1 (FHV-1) infection: a preliminary dose determination trial (abstr). Transactions Am Coll Vet Ophthalmol 1996;27:79.
Truyen U, Blewaska B, Schultheiss U. Untersuchungen der antiviralen Wirksamkeit von Interferon Omega gegen ausgewählte Viren von Hund und Katze. 47 (abstr), in Proceedings. Annu Conv Soc German Small Anim Vet and Conf Fed Eur Companion Anim Vet Assoc 2001. Available at: www.tgd.bayern.de. Accessed Jun 19, 2005.
Crandell Rees Feline Kidney Cells (CRFK), CCL-94, American Type Culture Collection, Manassas, Va.
Feline Herpesvirus 1 (strain C-27), VR-636, American Type Culture Collection, Manassas, Va.
Intron-A, Schering Corp, Kenilworth, NJ.
Generously provided by Virbagen Omega, Virbac SA, Carros, France.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
JMP statistical software, SAS Institute Inc, Cary, NC.
Verneuil M. Topical application of feline interferon omega in the treatment of herpetic keratitis in the cat (abstr). Vet Ophthalmol 2004;7:427.
Bräcklein T, Speier S, Metzler A, et al. Activity of topically and orally applied interferon omega in cats indicated by Mx-protein expression in conjunctival and white blood cells (abstr). Vet Ophthalmol 2003;6:358.
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