BACKGROUND
feline herpesvirus  <dig> , an alphaherpesvirus, is one of the most common viruses among cats  <cit> .
infection is associated with respiratory disorders and ocular disease, including keratitis, conjunctivitis, corneal sequestration, keratoconjunctivitis and, ultimately, loss of sight  <cit> .
the severity of clinical symptoms induced by fhv- <dig> infection can be reduced by vaccination  <cit> .
however, especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may still occur.
once these kittens have developed lesions, recurrent disease and virus excretion may occur upon reactivation at later age  <cit> .
in order to control disease in these cases, treatment with an effective antiviral drug would be helpful.
several antiviral drugs have already been tested for their efficacy to inhibit fhv- <dig> replication by means of a classical plaque number reduction assay.
based on the ic <dig> or concentration needed to reduce plaque number with  <dig> %, especially the acyclic nucleoside phosphonate  -9-adenine   <cit>  seems very efficient.
also, the anp cidofovir  <cit>  as well as the nucleoside analogues ganciclovir, penciclovir  <cit> , idoxuridine  <cit> , trifluridine  <cit>  and vidarabine  <cit>  are efficient in reducing the number of fhv-1-induced plaques in vitro.
acyclovir and foscarnet, although of proven value against human herpesviruses, have low efficacy against the formation of plaques by fhv- <dig>  <cit> .
besides by reducing the number of plaques, as described above, antiviral agents may also exert an effect by reducing the size of herpesvirus-induced plaques  <cit> .
reduction in plaque size may be a potential parameter for the ability of an antiviral agent to restrict the size of macroscopic lesions in vivo  <cit> .
up till now, no studies have addressed the efficacy of antiviral agents to limit the size of fhv-1-induced plaques.
the aim of the present study was to compare the efficacy of six antiviral drugs against fhv- <dig> in vitro.
parameters used to measure efficacy were the ability to limit fhv-1-induced plaque number ) as well as plaque size ) in crandell-rees feline kidney  cells.
RESULTS
effect of the antiviral drugs on plaque number
no significant variation in plaque number was observed between fhv-1-inoculated cells incubated with various concentrations of roswell park memorial institute - <dig> medium and the untreated control .
effect of the antiviral drugs on plaque size
it is known that marked differences may occur in plaque size when fhv- <dig> 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 .
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 .
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 .
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 .
effect of the antiviral drugs on the viability of crfk cells
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 .
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 .
acyclovir exerted no significant effect on the viability of crfk cells up till a concentration of  <dig> μg/ml , ganciclovir up till a concentration of  <dig> μg/ml , cidofovir up till a concentration of  <dig> μg/ml , foscarnet up till a concentration of  <dig> μg/ml , adefovir up till a concentration of  <dig> μg/ml  and pmedap up till a concentration of  <dig> μg/ml .
these concentrations amply exceeded the ic <dig>  and the ic <dig>  for all drugs , demonstrating that none of the products were toxic for crfk cells at antiviral concentrations.
discussion
over the years, an impressive array of antiviral agents has been developed for the treatment of human herpesvirus infections  <cit> .
many of these agents have already been studied for their efficacy against fhv- <dig>  <cit> .
in the present study the efficacy of a selection of six antiviral drugs against fhv- <dig> was compared in crfk cells using two different parameters, i.e.
reduction in plaque number and reduction in plaque size.
our study demonstrates that the efficacy of the drugs to reduce the number of plaques can be ranked as follows: ganciclovir  → pmedap  → cidofovir  → adefovir  → foscarnet  → acyclovir .
the results for ganciclovir and cidofovir are similar to those obtained by maggs and clarke  <cit> , who compared the ability of four antiviral drugs to reduce the number of plaques in crfk cells.
they found an ic <dig>  of  <dig>  μm for ganciclovir and of  <dig> μm for cidovir .
concerning the activity of foscarnet and acyclovir, our results are slightly different from those obtained by maggs and clarke  <cit> .
the latter authors found a lower activity for foscarnet   <dig>  μm or relative antiviral efficacy  <dig> ) and a higher activity for acyclovir   <dig>  μm or relative antiviral efficacy  <dig> ).
also, other authors obtained variable results on the activity of acyclovir against fhv- <dig>  with ic <dig>  values ranging from  <dig>  to  <dig> μm  <cit> .
a potential explanation for the observed variation may be related to the virus strain.
indeed, in a comparative study of nasisse et al.
<cit> , the ic <dig>  for acyclovir in crfk cells varied almost 2-fold depending on the fhv- <dig> strain used.
also, there may be inter-test variation involved.
while both testing the activity of acyclovir against fhv- <dig> strain  <dig> on crfk cells, nasisse et al.
<cit>  reported a markedly higher ic <dig>   than maggs and clarke  <cit>  .
antiviral agents not only reduce the number of herpesvirus-induced plaques, they also contribute in the reduction of plaque size  <cit> .
our study is the first that addressed the ability of antiviral agents to reduce the size of fhv-1-induced plaques.
ranking the drugs following their relative ability to reduce plaque size resulted in cidofovir  → ganciclovir  → pmedap  → adefovir  → foscarnet  → acyclovir , which is an almost exactly equivalent ranking as that for plaque number data.
the latter demonstrates that measuring plaque size is a useful and complementary means of assessing antiviral efficacy.
interestingly, for all six drugs it was found that the concentration needed to reduce plaque size was notably lower than that needed to reduce plaque number .
the most remarkable effect was observed for cidofovir.
its ic <dig>  was 30-times lower than its ic <dig> .
for ganciclovir, the ic <dig>  was 8-times lower.
for the other four drugs, a  <dig> to 5-times lower concentration was required to reduce plaque size with 50% when compared with the ic <dig> .
the relevance of this finding in view of protection of cats against fhv-1-induced disease remains to be determined.
however, as already speculated by mikloska and cunningham  <cit> , reduction of plaque size in vitro may be a potential parameter for the ability of an antiviral agent to restrict the size of virus-induced lesions in vivo.
the ability of adefovir to inhibit fhv- <dig> replication was found to differ markedly from pmedap.
this seems rather surprising since both drugs, which belong to the class of acyclic nucleoside phosphonates, are closely related and share a similar antiviral mechanism that relies on the inhibition of viral dna polymerase by the corresponding active diphosphate derivatives.
a possible explanation could be a difference in uptake of the drugs by crfk cells.
however, kramata and downey  <cit>  found that cellular uptake did not significantly differ between adefovir and pmedap in a human t-lymphoblastoid cell line ccrf-cem.
alternatively, viral dna polymerase may have a higher sensitivity for the pmedap diphosphate derivatives than for adefovir diphosphate derivatives, similar as described for cellular dna polymerase  <cit> .
also, there may be a variation in intracellular stability of each drug metabolite as observed for other antiviral drugs  <cit> .
it seems unlikely that the efficiency of phosphorylation by cellular enzymes and, consequently, the amounts of the active diphosphates differ.
for both drugs, phosphorylation is catalyzed by mitochondrial and cytosolic isoenzymes of amp kinase  <cit> .
none of the products were toxic for crfk cells at antiviral concentrations.
however, the results of the cytotoxicity assay cannot simply be extrapolated to the cat itself, as sensitivity of host cells to a certain drug may differ from those of a continuous cell line.
for example, while we were unable to detect a significant effect of cidofovir on the viability of crfk cells up to a concentration of  <dig> μg/ml, sandmeyer and colleagues  <cit>  reported cytotoxic effects at a concentration of  <dig> μg/ml in a primary cell culture of feline corneal epithelial cells.
also, adefovir exerted no cytotoxic effects in our in vitro study, but when applied in cats for the treatment of feline infectious peritonitis virus  <cit>  or feline leukaemia virus  <cit> , severe haematological side effects were observed.
this highlights the need for extensive safety studies in the cat.
CONCLUSIONS
from our study, it can be concluded that measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug.
by using these parameters for six selected antiviral drugs, we found that ganciclovir, pmedap, and cidofovir are most potent inhibitors of fhv- <dig> replication in crfk cells.
therefore, they may be valuable candidates for the treatment of fhv- <dig> infection in cats.

 feline herpesvirus, an alphaherpesvirus, is one of the most common viruses among cats. infection is associated with respiratory disorders and ocular disease, including keratitis, conjunctivitis, corneal sequestration, keratoconjunctivitis and, ultimately, loss of sight. the severity of clinical symptoms induced by fhv- infection can be reduced by vaccination. however, especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may still occur. once these kittens have developed lesions, recurrent disease and virus excretion may occur upon reactivation at later age. in order to control disease in these cases, treatment with an effective antiviral drug would be helpful. several antiviral drugs have already been tested for their efficacy to inhibit fhv- replication by means of a classical plaque number reduction assay. based on the ic or concentration needed to reduce plaque number with %, especially the acyclic nucleoside phosphonate -9-adenine seems very efficient. also, the anp cidofovir as well as the nucleoside analogues ganciclovir, penciclovir, idoxuridine, trifluridine and vidarabine are efficient in reducing the number of fhv-1-induced plaques in vitro. acyclovir and foscarnet, although of proven value against human herpesviruses, have low efficacy against the formation of plaques by fhv-. besides by reducing the number of plaques, as described above, antiviral agents may also exert an effect by reducing the size of herpesvirus-induced plaques. reduction in plaque size may be a potential parameter for the ability of an antiviral agent to restrict the size of macroscopic lesions in vivo. up till now, no studies have addressed the efficacy of antiviral agents to limit the size of fhv-1-induced plaques. the aim of the present study was to compare the efficacy of six antiviral drugs against fhv- in vitro. parameters used to measure efficacy were the ability to limit fhv-1-induced plaque number ) as well as plaque size ) in crandell-rees feline kidney cells.  effect of the antiviral drugs on plaque number no significant variation in plaque number was observed between fhv-1-inoculated cells incubated with various concentrations of roswell park memorial institute - medium and the untreated control. effect of the antiviral drugs on plaque size it is known that marked differences may occur in plaque size when fhv- 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. 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. 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. 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. effect of the antiviral drugs on the viability of crfk cells 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. 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. acyclovir exerted no significant effect on the viability of crfk cells up till a concentration of μg/ml, ganciclovir up till a concentration of μg/ml, cidofovir up till a concentration of μg/ml, foscarnet up till a concentration of μg/ml, adefovir up till a concentration of μg/ml and pmedap up till a concentration of μg/ml. these concentrations amply exceeded the ic and the ic for all drugs, demonstrating that none of the products were toxic for crfk cells at antiviral concentrations. discussion over the years, an impressive array of antiviral agents has been developed for the treatment of human herpesvirus infections. many of these agents have already been studied for their efficacy against fhv-. in the present study the efficacy of a selection of six antiviral drugs against fhv- was compared in crfk cells using two different parameters, i.e. reduction in plaque number and reduction in plaque size. our study demonstrates that the efficacy of the drugs to reduce the number of plaques can be ranked as follows: ganciclovir → pmedap → cidofovir → adefovir → foscarnet → acyclovir. the results for ganciclovir and cidofovir are similar to those obtained by maggs and clarke, who compared the ability of four antiviral drugs to reduce the number of plaques in crfk cells. they found an ic of μm for ganciclovir and of μm for cidovir. concerning the activity of foscarnet and acyclovir, our results are slightly different from those obtained by maggs and clarke. the latter authors found a lower activity for foscarnet μm or relative antiviral efficacy ) and a higher activity for acyclovir μm or relative antiviral efficacy ). also, other authors obtained variable results on the activity of acyclovir against fhv- with ic values ranging from to μm. a potential explanation for the observed variation may be related to the virus strain. indeed, in a comparative study of nasisse et al., the ic for acyclovir in crfk cells varied almost 2-fold depending on the fhv- strain used. also, there may be inter-test variation involved. while both testing the activity of acyclovir against fhv- strain on crfk cells, nasisse et al. reported a markedly higher ic than maggs and clarke. antiviral agents not only reduce the number of herpesvirus-induced plaques, they also contribute in the reduction of plaque size. our study is the first that addressed the ability of antiviral agents to reduce the size of fhv-1-induced plaques. ranking the drugs following their relative ability to reduce plaque size resulted in cidofovir → ganciclovir → pmedap → adefovir → foscarnet → acyclovir, which is an almost exactly equivalent ranking as that for plaque number data. the latter demonstrates that measuring plaque size is a useful and complementary means of assessing antiviral efficacy. interestingly, for all six drugs it was found that the concentration needed to reduce plaque size was notably lower than that needed to reduce plaque number. the most remarkable effect was observed for cidofovir. its ic was 30-times lower than its ic. for ganciclovir, the ic was 8-times lower. for the other four drugs, a to 5-times lower concentration was required to reduce plaque size with 50% when compared with the ic. the relevance of this finding in view of protection of cats against fhv-1-induced disease remains to be determined. however, as already speculated by mikloska and cunningham, reduction of plaque size in vitro may be a potential parameter for the ability of an antiviral agent to restrict the size of virus-induced lesions in vivo. the ability of adefovir to inhibit fhv- replication was found to differ markedly from pmedap. this seems rather surprising since both drugs, which belong to the class of acyclic nucleoside phosphonates, are closely related and share a similar antiviral mechanism that relies on the inhibition of viral dna polymerase by the corresponding active diphosphate derivatives. a possible explanation could be a difference in uptake of the drugs by crfk cells. however, kramata and downey found that cellular uptake did not significantly differ between adefovir and pmedap in a human t-lymphoblastoid cell line ccrf-cem. alternatively, viral dna polymerase may have a higher sensitivity for the pmedap diphosphate derivatives than for adefovir diphosphate derivatives, similar as described for cellular dna polymerase. also, there may be a variation in intracellular stability of each drug metabolite as observed for other antiviral drugs. it seems unlikely that the efficiency of phosphorylation by cellular enzymes and, consequently, the amounts of the active diphosphates differ. for both drugs, phosphorylation is catalyzed by mitochondrial and cytosolic isoenzymes of amp kinase. none of the products were toxic for crfk cells at antiviral concentrations. however, the results of the cytotoxicity assay cannot simply be extrapolated to the cat itself, as sensitivity of host cells to a certain drug may differ from those of a continuous cell line. for example, while we were unable to detect a significant effect of cidofovir on the viability of crfk cells up to a concentration of μg/ml, sandmeyer and colleagues reported cytotoxic effects at a concentration of μg/ml in a primary cell culture of feline corneal epithelial cells. also, adefovir exerted no cytotoxic effects in our in vitro study, but when applied in cats for the treatment of feline infectious peritonitis virus or feline leukaemia virus, severe haematological side effects were observed. this highlights the need for extensive safety studies in the cat.  from our study, it can be concluded that measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. by using these parameters for six selected antiviral drugs, we found that ganciclovir, pmedap, and cidofovir are most potent inhibitors of fhv- replication in crfk cells. therefore, they may be valuable candidates for the treatment of fhv- infection in cats. 
the results demonstrate that the ic values were notably lower than the ic values. feline herpesvirus is a common cause of respiratory and ocular disease in cats. the most remarkable effect was observed for cidofovir and ganciclovir. especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may occur. therefore, they may be valuable candidates for the treatment of fhv- infection in cats. by using these parameters for six selected antiviral drugs, we found that ganciclovir, pmedap, and cidofovir are the most potent inhibitors of fhv- replication in crfk cells. in the present study, the efficacy of six antiviral drugs, i.e. all antiviral drugs were able to significantly reduce plaque size when compared with the untreated control. in conclusion, measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. none of the products were toxic for crfk cells at antiviral concentrations. acyclovir, ganciclovir, cidofovir, foscarnet, adefovir and 9-- 6-diaminopurine, against fhv- was compared in crandell-rees feline kidney cells using reduction in plaque number and plaque size as parameters. as observed for the reduction in plaque number, ganciclovir, pmedap and cidofovir were most potent in reducing plaque size.
 feline herpesvirus is a common cause of respiratory and ocular disease in cats. especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may occur. therefore, there is a need for an effective antiviral treatment. in the present study, the efficacy of six antiviral drugs, i.e. acyclovir, ganciclovir, cidofovir, foscarnet, adefovir and 9-- 6-diaminopurine, against fhv- was compared in crandell-rees feline kidney cells using reduction in plaque number and plaque size as parameters.  the capacity to reduce the number of plaques was most pronounced for ganciclovir, pmedap and cidofovir. ic values were μg/ml, μg/ml and μg/ml, respectively. adefovir and foscarnet were intermediately efficient with an ic of μg/ml and μg/ml, respectively. acyclovir was least efficient of μg/ml or μm). all antiviral drugs were able to significantly reduce plaque size when compared with the untreated control. as observed for the reduction in plaque number, ganciclovir, pmedap and cidofovir were most potent in reducing plaque size. ic values were μg/ml, μg/ml and μg/ml, respectively. adefovir and foscarnet were intermediately potent, with an ic of μg/ml and μg/ml, respectively. acyclovir was least potent of μg/ml or μm). the results demonstrate that the ic values were notably lower than the ic values. the most remarkable effect was observed for cidofovir and ganciclovir. none of the products were toxic for crfk cells at antiviral concentrations.  in conclusion, measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. by using these parameters for six selected antiviral drugs, we found that ganciclovir, pmedap, and cidofovir are the most potent inhibitors of fhv- replication in crfk cells. therefore, they may be valuable candidates for the treatment of fhv- infection in cats. 