Antiherpes activity of the immunomodulator OK-432, a streptococcal preparation, in immunosuppressed mice

The antiviral activity of OK-432, an antitumor agent originating from Streptococcal preparations, against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). Intraperitoneal administration of OK-432 to mice 1 day after treatment with 200 mg CY/kg prevented death due to HSV-2 encephalitis in a dose-dependent manner. When the immunosuppressed mice were given OK-432 prior to HSV-2 infection, both by the intraperitoneal route, virus growth in the peritoneal cavity was significantly suppressed. Following with OK-432, the number of macrophages in immunosuppressed mice was increased to a significantly greater extent than the numbers of lymphocytes and polymorphonuclear leukocytes. The intrinsic antiviral activity of macrophages against HSV-2 as well as the natural killer (NK) activity against YAC-1 target cells was significantly enhanced by OK-432 in immunosuppressed mice.     

Enhancement of nonspecific resistance to viral infection by chemically synthesized lipid A-subunit analogs with different backbone structures and acyl groups

Protection against vaccinia virus infection and induction of interferon (IFN) were investigated in Propionibacterium acnes-primed mice following treatment with chemically synthesized lipid A-subunit derivatives. The antiviral activity was based on the reduction of numbers of tail lesions in mice injected intravenously with the test compounds 1 day before virus infection. GLA-27, a 4-O-phosphono-D-glucosamine carrying 3-O-tetradecanoyl (C14) and N-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups, offered significant antiviral activity. Chemical modifications at the C1 position of GLA-27, e.g. phosphorylation, replacement of OH by an SH, did not cause a significant change in antiviral activity. GLA-57 carrying an N-3-dodecanoyloxytetradecanoyl group showed stronger activity than GLA-27, but GLA-58 carrying an N-3-hexadecanoyloxytetradecanoyl group did not exhibit significant activity. GLA-59 carrying 3-O-3-hydroxytetradecanoyl and N-C14-O-(C14) groups was more active than GLA-27 and GLA-57. GLA-60 possessing the same fatty acid substituents as GLA-59 but in the reversed order was the most active of all compounds tested. This suggests that the nature and position of the acyl substituents are important for achieving the antiviral effects. The (R) isomers of GLA-59 and GLA-60 possessed stronger IFN-inducing activity than the (S) isomers, but no significant difference in antiviral activity was seen between the isomers.     

Antiviral effect of aqueous extracts from species of the Lamiaceae family against Herpes simplex virus type 1 and type 2 in vitro

Aqueous extracts from species of the Lamiaceae family were examined for their antiviral activity against Herpes simplex virus (HSV). Extracts from lemon balm (Melissa officinalis), peppermint (Mentha x piperita), prunella (Prunella vulgaris), rosemary (Rosmarinus officinalis), sage (Salvia officinalis) and thyme (Thymus vulgaris) were screened. Their inhibitory activity against Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and an acyclovir-resistant strain of HSV-1 (ACV (res)) was tested in vitro on RC-37 cells in a plaque reduction assay. The 50% inhibitory concentrations (IC (50)) of the extracts for HSV plaque formation were determined in dose-response studies. All test compounds showed a high antiviral activity against HSV-1, HSV-2 and ACV (res). In order to identify the mode of antiviral action, the extracts were added to the cells or viruses at different stages of infection. Both types of Herpes virus including ACV (res) were considerably neutralized after treatment with the extracts prior to infection. At maximum non-cytotoxic concentrations of the extracts, plaque formation was significantly reduced by > 90% for HSV-1 and HSV-2 and > 85% for ACV (res). In time-response studies over a period of 2 hours, a clearly time-dependent activity was demonstrated. These results indicate that the extracts affect HSV before adsorption, but have no effect on the intracellular virus replication. Therefore, the extracts exert their antiviral effect on free HSV and offer a chance to use them for topical therapeutic application against recurrent HERPES infections.     

A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1) infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice against HSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity in a phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of both HSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should be welcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug bound by a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase their phagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protection against both HSV-1 and HIV-1 replication in human macrophages.     

Antiviral and immunomodulating activities of chemically synthesized lipid A-subunit analogues GLA-27 and GLA-60

Biological and antiviral activities of chemically synthesized lipid A-subunit analogues, GLA-27 and GLA-60, were investigated with respect to defense mechanisms such as macrophage and natural killer (NK) cell activation and interferon (IFN)-inducing activity. GLA-27, a 4-O-phosphono-D-glucosamine derivative carrying 3-O-tetradecanoyl (C14) and 2-N-3-tetradecanoyloxytetradecanoyl (C14-O-(C14] group, and GLA-60, a similar analogue carrying 3-O-linked C14-O-(C14) and 2-N-linked 3-hydroxytetradecanoyl (C14-OH) groups, strongly inhibited the formation of pox tail lesions and the growth of vaccinia virus at the tail lesion sites in infected mice. The antiviral activity of GLA-60 was about 1000-fold higher than that of muramyldipeptide (MDP), a representative immunomodulator. GLA-27 and GLA-60 had stronger immunomodulating activity than MDP in macrophage activation, NK cell activation and IFN-inducing activity, although it was weaker than natural lipid A. Toxic manifestations such as pyrogenicity, local Schwartzman reaction and lethality were far less pronounced for GLA-27 and GLA-60 than for natural lipid A.     

Potentiating effect of ribavirin on the anti-herpes activity of acyclovir.

The combined antiviral effects of acyclovir (ACV) and ribavirin (Rbv) on herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) in cell cultures and on experimental HSV-1 keratitis in rabbits were studied. The antiviral activity in vitro was based on cytopathogenicity inhibition and yield reduction. The combination of the two drugs exhibited synergy as evaluated graphically (isobolograms). Rbv also potentiated the antiviral effect of ACV in vivo, in the experimental HSV-1 keratitis model in rabbits. This was evident from both the severity of corneal lesions and virus shedding in the tear film. The potentiating effect of Rbv on the anti-HSV-1 activity of ACV in vitro was reversed by guanosine.     

Activity of two synthetic amphiphilic peptides and magainin-2 against herpes simplex virus types 1 and 2

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin-1 (mod-1) and modelin-5 (mod-5), and of the natural antibacterial peptide magainin-2 (mag-2) against herpes simplex viruses type 1 (HSV-1) and 2 (HSV-2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod- displayed a strong antiviral effect against HSV-1 and HSV-2, with 50% effective dose (ED₅₀) values of 4.6 and 4.1 μg/mL, respectively. Mag-2, mod-5 and a mixture of both had no significant inhibitory effect. Addition of mod-1 up to a concentration of 100μg/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue-exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod-1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod-1 may be an effective topical antiviral agent against herpes viruses.     

Lactoferrin and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir

Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Upon peptic digestion of LF, a peptide called lactoferricin (Lfcin) is generated. Lfcin corresponds to the N-terminal part of the protein. In this study we investigated the antiviral activity of bovine and human Lfcin against Herpes simplex virus (HSV)-1 and HSV-2. The 50% effective concentrations (EC(50)) for LF and Lfcin against several clinical isolates of HSV-1 and HSV-2, including acyclovir (ACV)-resistant strains, were determined. We further evaluated the effect of the combination of either LF or Lfcin with ACV against HSV-1 and HSV-2. Synergy was observed between both LF or Lfcin in combination with ACV against the HSV laboratory strains.The 50% effective concentration (EC(50)) for ACV and LF or Lfcin, when combined with ACV, could be reduced by two- to sevenfold compared to the EC(50) when the drugs were used alone.     

Acyclovir treatment of experimental genital herpes simplex virus infections. I. Topical therapy of type 2 and type 1 infections of mice

Intravaginal inoculation of mice with herpes simplex virus (HSV) provides a model infection of genital herpes to determine the effectiveness of potential antiviral agents. topical (intravaginal) treatment with 1% or 5% acyclovir (ACV) in an ointment of gel vehicle initiated 3, 6 or 24 h after inoculation with HSV type 2, significantly inhibited viral replication in the genital tract and usually reduced final mortality. Treatment with 5% ACV initiated 48 or 72 h after infection also reduced vaginal virus titers but did not alter final mortality. When mice were inoculated with HSV type 1 treatment with 5% ACV significantly reduced viral replication in the genital tract when begun as late as 72 h. In HSV-2 infected mice, treatment initiated 3 h but not 24 h after infection prevented the establishment of latent infection in sacral ganglie. These results suggest that topical ACV may be effective antiviral agent for primary genital herpes in humans.     

The synergistic effects of betulin with acyclovir against herpes simplex viruses

Betulin, a pentacyclic triterpenoid, was isolated from the bark of Betula papyrifera. The antiviral efficacies of betulin on herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were evaluated using viral plaque reduction assays on Vero cells. The results indicate that betulin is active against both HSV-1 and HSV-2 infections with the 50% effective concentrations (EC(50)) of 0.40 and 4.15 microg/ml, respectively. The cytotoxicity of betulin was examined on Vero cells using a neutral red uptake assay. The 50% cytotoxic concentration (CC(50)) of betulin was 73.1 microg/ml. A synergistic antiviral effect between betulin and acyclovir (ACV) was determined by drug combination studies. Strong and moderate synergistic antiviral effects were observed for betulin and ACV against HSV-1 when the concentrations of ACV and betulin were higher than 0.068 and 0.4 microg/ml, respectively. At the concentrations lower than these, additive effect was found. Synergistic antiviral effects were also found against HSV-2 at higher concentrations than for HSV-1, i.e. 0.45 microg/ml of ACV combined with 8.4 microg/ml of betulin.     

Combination of benzo[a]phenothiazines with acyclovir against herpes simplex virus.

The combined antiviral effects of some benzo[a]phenothiazines and 9-[2-hydroxy(ethoxy)methyl]guanine (acycloguanosine, acyclovir, ACV) on the multiplication of herpes simplex virus type 2 (HSV-2) were studied using Vero cells. The antiviral effect of ACV on a wild strain of HSV-2 was enhanced in the presence of 5-oxo-5H-benzo[a]phenothiazine and 6-methyl-5-oxo-5H-benzo[a]phenothiazine in a yield reduction test. A mathematical formula was used to interpret the drug interaction and a synergistic effect was found with a combination of ACV and benzo[a]phenothiazines. The effect of simultaneous application of two benzo[a]phenothiazines on the multiplication of HSV-2 strain during serial passages was also investigated. The combinations of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine with ACV at a low concentration using serial passages of a plaque-purified ACV sensitive HSV-2 strain, reduced the infective virus population. A similar effect was also found on the activity of other benzo[a]phenothiazine derivatives. When the two most effective derivatives of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine were simultaneously used with ACV against a wild type HSV-2 strain during consecutive passages, the infective virus titres were decreased, but their effect was only moderate. These results suggest that a combination of some benzo[a]phenothiazines with ACV might enhance their antiviral activity probably by reduction of the mutagenic rate in the virus populations.     

Enhancement of non-specific resistance to viral infection by muramyldipeptide and its analogs

Antiviral activity of muramyldipeptide (MDP) and its lipophilic derivatives, B30-MDP and MDP-Lys(L18), was investigated in mice infected with vaccinia virus (VV) and herpes simplex virus type 2 (HSV-2). Mice administered these compounds subcutaneously or orally were protected against VV in tail lesion tests and against HSV-2 in skin lesion tests, respectively. Since in vitro antiviral activity was not demonstrated with these compounds in cultured mammalian cells infected with either VV or HSV-2, host-mediated defense mechanisms may play a role in the activity of the compounds. As for serum interferon (IFN) induction, MDP and its analogs showed no activity in mice, suggesting that IFN does not participate in the antiviral mechanisms against VV and HSV-2. An extrinsic antiviral activity was demonstrated when peritoneal macrophages from the mice administered these compounds were cocultivated with VV-infected 3T3 cells. The results indicate that macrophage activation by MDP and its analogs plays a role in the defense mechanisms against viral infection. This activity was not virus-specific. We also demonstrate that the introduction of lipophilic residue(s) into MDP enhances the antiviral activity of mice against VV and HSV-2.     

Effect of Azone upon the in vivo antiviral efficacy of cidofovir or acyclovir topical formulations in treatment/prevention of cutaneous HSV-1 infections and its correlation with skin target site free drug concentration in hairless mice

The purpose of this study is to examine the influence of Azone upon the skin target site free drug concentration (C(*)) and its correlation with the in vivo antiviral efficacies of cidofovir (HPMPC) and acyclovir (ACV) against HSV-1 infections. Formulations of HPMPC and ACV with or without Azone were used. The in vitro skin flux experiments were performed and the C(*) values were calculated. For the in vivo efficacy studies, hairless mice cutaneously infected with HSV-1 were used and three different treatment protocols were carried out. The protocols were chosen based upon when therapy is initiated and terminated in such a way to assess the efficacy of the test drug to cure and/or prevent HSV-1 infections. A finite dose of the formulation was topically applied twice a day for the predetermined time course for each protocol and the lesions were scored on the fifth day. For ACV formulation with Azone, the C(*) values and hence the in vivo efficacy were much higher than those for that without Azone. In protocol #1, however, early treatment did not increase the in vivo efficacy of ACV when compared with the standard treatment protocol #3. In protocol #2 where the treatment was terminated on the day of virus inoculation, the efficacies for both ACV formulations were completely absent. Although the estimated C(*) values for HPMPC formulations with and without Azone were comparable, formulation with Azone was much more effective than that without Azone in all treatment protocols. HPMPC formulations with Azone at similar flux values were much more effective in "treating and preventing" HSV-1 infections than those without Azone. For ACV formulations, in contrast, addition of Azone has failed to show any effect on the preventive in vivo antiviral efficacy and the enhancement of ACV in vivo antiviral efficacy was merely the skin permeation enhancement effect of Azone.     

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)-cyclobutyl] guanine [(+-)-BHCG or SQ 33,054]: a potent and selective inhibitor of herpesviruses

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine [(+-)-BHCG or SQ 33,054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (+-)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (+-)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (+-)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.     

Effect of acyl substituents of synthetic lipid A-subunit analogues on their immunomodulating antiviral activity.

A chemically synthesized lipid A-subunit analogue, GLA-60, 2-deoxy-4-O-phosphono-2-[(3R)-3-hydroxytetradecanamido]-3-O-[(3R)- 3- tetradecanoyloxytetradecanoyl]-D-glucose, has many of the activities of endotoxin but has little toxicity. Then, compounds with various lengths of acyl side chain of the acyloxyacyl group at the 3-O position of GLA-60 were synthesized and evaluated for interferon (IFN)-inducing activity, natural killer (NK) cell activation and antiviral activity. The compounds with acyl side chains between C8 and C15 exhibited significant antiviral activity (inhibition of pox tail lesion formation in vaccinia virus-infected mice), serum IFN-inducing activity and NK cell activation. However, the compound carrying a C2 or a C16 acyl side chain did not exhibit these activities. The compounds with a C13 or C14 acyl side chain showed strong protective against herpes simplex virus type 1 in cyclophosphamide-immunosuppressed mice.     

(E)-5-(2-bromovinyl)-2'-desoxyuridine--a new nucleoside analog with selective inhibitory action against herpesviruses. Studies in cell culture and animal experiments

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (1; BrVUdR) inhibits the replication of herpes simplex virus type 1 (HSV-1) and of varicella-zoster virus (VZV) in vitro at concentrations of 0.01 to 0.23 mumol/l, whereas herpes simplex virus type 2 (HSV-2) is influenced only at 5.5 to 27 mumol/l. In comparison to some classical and newly developed antiherpetics, i. e. 5-iodo-2'-desoxyuridine (2; idoxuridine, IDU), 9-beta-D-arabinofuranosyladenine (4; vidarabine Ara-A), 9-(2-hydroxyethoxymethyl) guanine (5; acyclovir, ACV) and 2'-fluoro-5-iodo-1-beta-D-aracytosine (6;FIAC) the following order of decreasing activity was found:1 greater than 6 greater than 5 greater than 2 greater than 4 (against HSV-1) and 6 greater than 2 greater than 5 greater than 1 greater than 4 (against HSV-2). The high selectivity of the antiviral effect of BrVUdR towards HSV-1 and TZV is based on the fact, that proliferation of different mammalian cell lines is inhibited by 50% only at concentrations as high as 90 to 170 mumol/l, resulting in a therapeutical index of 1000 to 10,000. Successful treatment of an HSV-1 encephalitis in mice as well as an HSV-1 keratitis of rabbits confirmed the efficiency of 1 in experimental animal infections. No toxic side effects in both local and systemic applications were observed. Promising data from cell culture and animal experiments recommend 1 as a potential candidate for the local and systemic treatment of HSV-1 and VZV infections in man.     

Development of an aciclovir implant for the effective long-term control of herpes simplex virus type-1 infection in Vero cells and in experimentally infected SKH-1 mice

Human herpes simplex virus type-1 (HSV-1) is treatable with oral doses of an antiviral agent such as aciclovir (ACV), a drug that has poor bioavailability. An alternative for delivering ACV would employ a long-lived subcutaneous implant that would allow for near zero-order drug delivery kinetics. This study aimed to develop an implant composed of a matrix of silicone and ACV that is capable of sustained long-term release of ACV. Once the implants had been created, release of ACV from the implants was determined and quantified in vitro using a spectrophotometric assay for the drug. Solvent-exposed surface area of the implant (2.86 mm(2), 6.28 mm(2), 34.62 mm(2) and 100.48 mm(2)) had a significant effect on release kinetics, whereas temperature (37 degrees C, 25 degrees C and 4 degrees C) and pH (6.0, 7.0 and 8.0) did not. The implants were also used successfully to suppress HSV-1 (KOS)-induced cytopathic effect in cultured Vero cells. The implants protected HSV-1-infected SKH-1 mice from viral reactivation (n = 37; P = 0.0367) via ultraviolet light compared with mice that were untreated (n = 37). Furthermore, mice that received silicone-only implants had no lowered risk of reactivation (n = 34; P = 0.7268), demonstrating the antiviral efficacy of the ACV implants.     

Antiviral activity of the marine alga Symphyocladia latiuscula against herpes simplex virus (HSV-1) in vitro and its therapeutic efficacy against HSV-1 infection in mice

The antiviral activities of extracts from 5 species of marine algae collected at Haeundae (Pusan, Korea), were examined using plaque reduction assays. Although the activity of a methanol (MeOH) extract of Sargassum ringoldianum (Sargassaceae) was the most potent against several types of viruses, it was also cytotoxic. A MeOH extract of Symphyocladia latiuscula (Rhodomelaceae) and its fractions exhibited antiviral activities against acyclovir (ACV) and phosphonoacetic acid (PAA)-resistant (AP(r)) herpes simplex type 1 (HSV-1), thymidine kinase (TK(-)) deficient HSV-1 and wild type HSV-1 in vitro without cytotoxicity. The major component, 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) of a CH(2)Cl(2)-soluble fraction was active against wild type HSV-1, as well as AP(r) HSV-1 and TK(-) HSV-1 (IC(50) values of 5.48, 4.81 and 23.3 microg/ml, respectively). The therapeutic effectiveness of the MeOH extract and TDB from S. latiuscula was further examined in BALB/c mice that were cutaneously infected with HSV-1 strain 7401H. Three daily oral administrations of the MeOH extract and TDB significantly delayed the appearance of score 2 skin lesions (local vesicles) and limited the development of further score 6 (mild zosteriform) lesions in infected mice without toxicity compared with controls. In addition, TDB suppressed virus yields in the brain and skin. Therefore TDB should be a promising anti HSV agent.     

Studies on the antiviral activity of 5′-amino-2′,5′-dideoxy-5-iodouridine (AIU) against herpes viruses in vivo and in vitro

5′-Amino-2′,5′-dideoxy-5-iodouridine (AIU) is known to have antiviral activity against herpes simplex virus type 1 (HSV-1) in cell cultures but is less potent than the parent compound 5-iodo-2′-deoxyuridine (IDU). Studies on its activity in vivo are limited. AIU showed antiviral effects in BHK cells against wild-type HSV-1 but not a thymidine kinase-negative (TK^?) mutant, indicating the importance of phosphorylation of the compound by HSV-1-induced thymidine kinase for antiviral effects. When cells were coinfected with the TK^? mutant and a wild-type TK⁺ strain, both strains were inhibited by AIU, suggesting that the failure to phosphorylate AIU accounts for the resistance of the TK^? strain alone. AIU failed to limit death after systemic HSV-1 infection of mice when the drug was administered parenterally or orally, although IDU in similar treatment regimens was effective. Tested for efficacy in a local HSV-1 skin infection of mice, topical AIU in a petrolatum ointment or dimethyl sulphoxide (DMSO) did not reduce titres of virus in the skin or modify the clinical course of infection, whereas topical application of IDU in DMSO caused a significant reduction in the titres of virus in the skin and reduced both the occurrence and the severity of lesions. When administered subcutaneously or orally AIU had a slight antiviral effect against HSV-1 infection in the skin of mice. Moreover, intraperitoneally (i.p.) administered AIU limited HSV-1 replication in peritoneal cells of i.p. infected mice, indicating that AIU is inherently antiviral in vivo. The poor antiviral activity of AIU in vivo compared with IDU is attributed to its lower antiviral potency as judged by its activity in cell cultures and its inability to enter neural tissue.     

Nucleoside pools of acyclovir-treated herpes simplex type 1 infected cells

Nucleoside pools of herpes simplex type 1 (HSV-1)-infected and uninfected African green monkey kidney (GMK) cells and human fetal lung fibroblasts (HL) have been analysed with high-performance liquid chromatography (HPLC). The only nucleosides found in measurable amounts were deoxythymidine (dThd) and adenosine (Ado). The dThd pool seemed to be greater in GMK cells than in HL cells. dThd was also the only nucleoside excreted into the medium. HSV-1 infection reduced the dThd concentration of GMK cells. Addition of acyclovir (ACV) to HSV-1-infected GMK cells inhibited virus replication. This resulted in a dThd concentration similar to that of uninfected GMK cells. dThd added to HSV-1-infected GMK and HL cells reduced the antiviral action of ACV but not that of phosphonoformic acid (PFA). ACV is known to be activated mainly by HSV-induced deoxythymidine kinase (dTK), an enzyme which utilizes dThd as a substrate, while the action of PFA is independent of dTK. The low antiviral activity of ACV in GMK cells as compared to HL cells may be explained by the presence of high amounts of dThd in GMK cells.