Figure 1 presents the dose-response curves regarding the inhibitory effect of each antiviral drug on plaque number (solid lines). The IC_{50 (NUMBER)} values were extrapolated from the curves and are presented in Table 1. The capacity to reduce the number of plaques was most pronounced for ganciclovir, PMEDAP and cidofovir. Adefovir and foscarnet were intermediately efficient. Acyclovir was least efficient.

No significant variation in plaque number was observed between FHV-1-inoculated cells incubated with various concentrations of Roswell Park Memorial Institute (RPMI)-1640 medium and the untreated control (LSD, 95% confidence interval).

It is known that marked differences may occur in plaque size when FHV-1 is grown in vitro. Therefore, data obtained in the plaque size assay were subjected to an F-test to examine whether the variation in plaque size between samples was significantly different from the variation in plaque size within a sample (95% confidence interval). Or, in other words, the F-test was used to answer the question whether variation in plaque size was merely an artefact or whether it was related to the use of antiviral drugs. It was found that all antiviral drugs exhibited a significant effect on plaque size (significance ≤ 0.001). Using a post-hoc LSD-test it was demonstrated that the reduction in plaque size was significant for all drugs at all concentrations when compared with the untreated control (95% confidence interval). The variation in plaque size observed in FHV-1-inoculated cells incubated with various concentrations of RPMI was not significantly different from the variation in plaque size observed in the untreated control (F-test, 95% confidence interval).

Figure 1 presents the dose-response curves regarding the inhibitory effect of each antiviral drug on plaque size (dashed lines). The IC_{50 (SIZE)} values were extrapolated from the curves and are presented in Table 1. As observed for the reduction in plaque number, ganciclovir, PMEDAP and cidofovir were most potent in reducing plaque size. Adefovir and foscarnet were intermediately potent and acyclovir was least potent. For all six drugs, the IC_{50 (SIZE)} was notably lower than the IC_{50 (NUMBER)} (Figure 1; Table 1). The most remarkable effect was observed for cidofovir and ganciclovir.

It was found that when CRFK cells were cultivated in the presence of 2% RPMI or more, that the relative viability of the cells was significantly lower in comparison with untreated CRFK cells incubated with complete medium (LSD, 95% confidence interval). In order to determine whether the antiviral products exerted a cytotoxic effect we, therefore, compared the mean relative viability of CRFK cells incubated with antiviral products with the mean relative viability of CRFK cells incubated with the corresponding concentration of RPMI (t-test, 95% confidence interval). Acyclovir exerted no significant effect on the viability of CRFK cells up till a concentration of 500 μg/ml (2220.2 μM), ganciclovir up till a concentration of 1000 μg/ml (3918.5 μM), cidofovir up till a concentration of 160 μg/ml (573.1 μM), foscarnet up till a concentration of 320 μg/ml (1666.7 μM), adefovir up till a concentration of 160 μg/ml (585.7 μM) and PMEDAP up till a concentration of 160 μg/ml (476.6 μM). These concentrations amply exceeded the IC_{100 (NUMBER)} and the IC_{100 (SIZE)} for all drugs (Figure 1), demonstrating that none of the products were toxic for CRFK cells at antiviral concentrations.

CRFK cells (CCL-94, ATCC, Manassas, VA, USA) were grown and maintained in complete medium consisting of Minimum Essential Medium (MEM) (Invitrogen, Merelbeke, Belgium) supplemented with 5 % fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.1 mg/ml kanamycin, 0.3 mg/ml glutamin and 1 mg/ml lactalbumin hydrolysate. Cells were trypsinized once a week.

The Belgian strain 94K49 was used for inoculation. This strain was isolated from a cat with respiratory disorders and identified as FHV-1 using monoclonal antibodies 22C12, 22F4 and 41G4 against FHV-1 glycoprotein gC, gB, and gD, respectively 26. Virus used for inoculation was at the 3^rd passage on CRFK cells.

Acyclovir, ganciclovir, adefovir and PMEDAP were dissolved in RPMI-1640 medium (Invitrogen, Merelbeke, Belgium) at a concentration of 1 mg/ml. Cidofovir and foscarnet were dissolved in RPMI-1640 at a concentration of 2 mg/ml. Subsequently, all drugs were filtered through a 0.45 μm filter to obtain a sterile solution and stored at 4°C. Before use, acyclovir was adapted to room temperature to re-dissolve possible precipitates.

CRFK-cells were grown to confluency in 24-well culture plates (Nunc A/S, Roskilde, Denmark) in complete medium at 37°C and 5 % CO₂. Then, medium was removed and cells in each well were inoculated with 40 PFU of FHV-1 in 200 μl of complete medium. After 1 h incubation at 37°C and 5 % CO₂, cells were washed twice with MEM. Afterwards, they were overlaid with medium containing carboxymethylcellulose (CMC) (0.94 %) and various concentrations of antiviral drugs. First, a broad range of concentrations was tested to allow a preliminary estimation of the antiviral efficacy. Then, final concentrations were chosen, as indicated in Figure 1. FHV-1-inoculated cells incubated with CMC and complete medium without antiviral drugs were included as an untreated control. Additionally, FHV-1-inoculated cells incubated with CMC and various concentrations of RPMI, ranging from 0.25 % to 20 % in complete medium, were included to test whether RPMI influenced plaque formation by FHV-1.

At 72 hours post inoculation, overlay medium was removed, cells were rinsed with PBS and, consecutively, fixed with paraformaldehyde 4 % and methanol + 1 % H₂O₂. An immunoperoxidase monolayer assay (IPMA) was performed based on van der Meulen et al. 27. Briefly, cells were incubated with a mixture of monoclonal antibodies 22C12, 22F4 and 41G4 against FHV-1 glycoprotein gC, gB, and gD, respectively 26 for 1 h at 37°C. After three washing steps, peroxidase-labelled goat anti-mouse IgG (Molecular Probes) were added for 1 h at 37°C. Following three additional washing steps, substrate solution of 3-amino-9-ethylcarbazole in 0.05 M acetate buffer with 0.05 % hydrogen peroxide was added. Cells were incubated for 10 minutes at 37°C and then, substrate solution was replaced with acetate buffer to block enzymatic staining.

The number of FHV-1-induced plaques was counted for each concentration of antiviral drug, for each concentration of RPMI and for untreated control samples using light microscopy (Olympus IX50). The inhibitory effect of the antiviral drugs on plaque number was calculated by following formula:

Percentage inhibition ( NUMBER ) = ( 1 − ( p l a q u e   n u m b e r ) a n t i v i r a l ( p l a q u e   n u m b e r ) c o n t r o l ) × 100 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqqGqbaucqqGLbqzcqqGYbGCcqqGJbWycqqGLbqzcqqGUbGBcqqG0baDcqqGHbqycqqGNbWzcqqGLbqzcqqGGaaicqqGPbqAcqqGUbGBcqqGObaAcqqGPbqAcqqGIbGycqqGPbqAcqqG0baDcqqGPbqAcqqGVbWBcqqGUbGBdaWgaaWcbaGaeiikaGIaeeOta4KaeeyvauLaeeyta0KaeeOqaiKaeeyrauKaeeOuaiLaeiykaKcabeaakiabg2da9iabcIcaOiabigdaXiabgkHiTmaalaaabaGaeiikaGIaemiCaaNaemiBaWMaemyyaeMaemyCaeNaemyDauNaemyzauMaeeiiaaIaemOBa4MaemyDauNaemyBa0MaemOyaiMaemyzauMaemOCaiNaeiykaKIaemyyaeMaemOBa4MaemiDaqNaemyAaKMaemODayNaemyAaKMaemOCaiNaemyyaeMaemiBaWgabaGaeiikaGIaemiCaaNaemiBaWMaemyyaeMaemyCaeNaemyDauNaemyzauMaeeiiaaIaemOBa4MaemyDauNaemyBa0MaemOyaiMaemyzauMaemOCaiNaeiykaKIaem4yamMaem4Ba8MaemOBa4MaemiDaqNaemOCaiNaem4Ba8MaemiBaWgaaiabcMcaPiabgEna0kabigdaXiabicdaWiabicdaWaaa@96B5@

The minimal concentration of each product required to reduce the plaque number with 50 % (IC_{50 (NUMBER)}) was hand-calculated from the dose-response curves generated from the data.

The size of FHV-1-induced plaques for each concentration of antiviral drug, for each concentration of RPMI and for untreated control samples was determined as follows. Plaques were photographed using light microscopy (Olympus IX50) and a digital camera (Sony Progressive 3CCD), attached to a Macintosh computer. Plaques were randomly selected in each sample and the number counted varied between a minimum of 3 and a maximum of 17 (mean number counted per sample was 10.4 ± 2.5). Then, the size of each of the plaques was determined in pixels using Scion 1.63 (National Institutes of Health) and, finally, the mean size per sample was calculated. The inhibitory effect of the antiviral drugs on plaque size was subsequently calculated by following formula:

Percentage inhibition ( SIZE ) = ( 1 − ( p l a q u e   s i z e ) a n t i v i r a l ( p l a q u e   s i z e ) c o n t r o l ) × 100 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=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@8F2F@

The minimal concentration of each product required to reduce the plaque size with 50 % (IC_{50 (SIZE)}) was hand-calculated from the dose-response curves generated from the data.

For each experiment, three independent replicates were performed.

In order to determine whether the antiviral drugs exerted a toxic effect on CFRK cells, a neutral red assay was performed. This assay is based on the incorporation of the supravital dye neutral red into living cells. In brief, CRFK-cells were grown to confluency in 96-well culture plates (Nunc A/S, Roskilde, Denmark) in complete medium at 37°C and 5 % CO₂. Then, medium was removed, cells were incubated with various concentrations of antiviral drugs ranging from 5 to 500 μg/ml in medium and cultured for 72 h at 37°C and 5 % CO₂. Cells incubated for 72 h with complete medium lacking antiviral drugs were included as viable controls. Cells successively incubated with complete medium for 70 h and distilled water for 2 h were included as non-viable controls. Additionally, cells incubated for 72 h with various concentrations of RPMI, ranging from 0.25% to 50% in complete medium, were included to test whether RPMI influenced the viability of CRFK cells.

After incubation, 50 μl of neutral red solution (0.1% in distilled water) was added to each well and plates were incubated at 37°C and 5% CO₂ for 1 h. Then, cells were rinsed with phosphate-buffered salt solution and air-dried at room temperature for 1 h. Finally, 50 μl of SDS (10% in distilled water) and 100 μl HC1 (0.2 M) were added and plates were shaken for 15 sec. Absorbance was measured on a Multiskan RC (Thermo Labsystems) using a 492-nm filter. Viability of cells was calculated by following formula:

Percentage of viable cells = O D t − O D d O D c − O D d × 100 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqqGqbaucqqGLbqzcqqGYbGCcqqGJbWycqqGLbqzcqqGUbGBcqqG0baDcqqGHbqycqqGNbWzcqqGLbqzcqqGGaaicqqGVbWBcqqGMbGzcqqGGaaicqqG2bGDcqqGPbqAcqqGHbqycqqGIbGycqqGSbaBcqqGLbqzcqqGGaaicqqGJbWycqqGLbqzcqqGSbaBcqqGSbaBcqqGZbWCcqGH9aqpdaWcaaqaaiabd+eapjabdseaejabdsha0jabgkHiTiabd+eapjabdseaejabdsgaKbqaaiabd+eapjabdseaejabdogaJjabgkHiTiabd+eapjabdseaejabdsgaKbaacqGHxdaTcqaIXaqmcqaIWaamcqaIWaamaaa@63E9@

where ODt is the absorbency of cells incubated with antiviral drugs or RPMI, ODd the absorbency of the non-viable cell control and ODc the absorbency of the viable cell control, respectively.

For each concentration of antiviral drug, for each concentration of RPMI and for control samples, eight wells were tested per experiment. Three independent replicates of each experiment were performed.

Statistical analysis was based on analysis of variance (ANOVA) using SPSS (SPSS Inc., Chicago, Illinois, USA).