Evaluation of dendrimer SPL7013, a lead microbicide candidate against herpes simplex viruses

Dendrimers are a novel class of polyanionic macromolecules with broad-spectrum antiviral activities and minimal toxicities. A new generation of amide dendrimer, SPL7013, was evaluated as a lead microbicide candidate against herpes simplex viruses (HSV). The plaque reduction assays showed that the 50% effective concentrations (EC(50)) determined by pre-treatment of cells were 2.0 microg/ml for HSV-1 and 0.5 microg/ml for HSV-2. Inhibitory effects were also observed on HSV-infected cells with EC(50)s of 6.1 microg/ml for HSV-1 and 3.8 microg/ml for HSV-2. These are the mean values from the test results of six batches of SPL7013. SPL7013 was also shown to be equally potent against HSV drug-resistant strains. SPL7013 completely inhibited viral adsorption to Vero cells at concentrations of higher than 3 microg/ml. Analyzed by a LightCycler assay after treatment of HSV-infected cells for 17 h, SPL7013 showed strong inhibition of HSV DNA synthesis with EC(50)s of approximately 6.2 and 2.0 microg/ml for HSV-1 and HSV-2, respectively. SPL7013 retained its anti-HSV activity even after treatment at acidic pHs 3.0 and 4.0 for 2 h. The presence of 10% human serum proteins did not affect the anti-HSV activity of SPL7013. SPL7013 was not toxic to Vero cells up to the highest concentration tested (10,000 microg/ml). Effects on cell proliferation were tested on two epithelial cell lines in both stationary and dividing phases. The 50% cytotoxic concentrations (CC(50)) in all cases were greater than 10,000 microg/ml. Our data indicate that SPL7013 is a promising candidate for development as a vaginal microbicide and a therapeutic agent.     

Evidence of dual sites of action of dendrimers: SPL-2999 inhibits both virus entry and late stages of herpes simplex virus replication

Dendrimers are macromolecules with broad-spectrum antiviral activity and minimal toxicity effective in animal models in preventing transmission of herpes simplex virus (HSV) infection. In order to further understand the mechanism of action, and toxicity profiles of the dendrimer SPL-2999 against HSV, we investigated in vitro activities as follows: modified plaque reduction assays for SPL-2999 showed that 50% effective concentrations (EC(50)) determined by pre-treatment of cells with SPL-2999 were 0.5 microg/ml (30 nM) for HSV-2 and 1 microg/ml (60 nM) for HSV-1, respectively. SPL-2999 was not toxic to Vero cells at concentration up to the highest tested (CC(50) greater than 1000 microg/ml). SPL-2999 appears to completely inhibit both viral adsorption and penetration to Vero cells at concentrations of higher than 3 microg/ml. Additionally, virus yield reduction assay showed that SPL-2999 was effective on cells already infected with HSV with EC(90)s (effective concentration giving 90% virus yield reduction) approximately 29.2 microg/ml for HSV-1 and 6.7 microg/ml for HSV-2. When Vero cells were infected with HSV at moi (multiplicity of infection) of 0.01 pfu/cell, the infected cells could be completely protected from viral cytopathic effect (CPE) by SPL-2999 with EC(90)s (effective concentration that protects 90% of cells from virus lysis) of 15 microg/ml for HSV-1 and 10 microg/ml for HSV-2. Results from Southern blot hybridization indicated that SPL-2999 inhibited DNA synthesis in HSV infected cells. We conclude that SPL-2999 inhibits both HSV entry into susceptible cells and late stages of HSV replication. Our data indicate that SPL-2999 is a potent inhibitor of both HSV-1 and -2 with the potential for further development as either a topical microbicide or a therapeutic agent.     

Inactivation of HSV-1 and HSV-2 and prevention of cell-to-cell virus spread by Santolina insularis essential oil

The essential oil obtained in toto from Santolina insularis was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The IC(50) values, determined by plaque reduction assays, were 0.88 and 0.7 microg/ml for HSV-1 and HSV-2, respectively, while the CC(50) determined by the MTT test on Vero cells was 112 microg/ml, indicating a CC(50)/IC(50) ratio of 127 for HSV-1 and 160 for HSV-2. Results obtained by plaque reduction assays also indicated that the antiviral activity of S. insularis was principally due to direct virucidal effects. Antiviral activity against HSV-1 and HSV-2 was not observed in a post-attachment assay, and attachment assays indicated that virus adsorption was not inhibited. Up to 80% inhibition of HSV-1 was achieved at the concentration of 40 microg/ml by yield reduction assay. Furthermore, reduction of plaque formation assays also showed that S. insularis essential oil inhibits cell-to-cell transmission of both HSV-1 and HSV-2.     

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.     

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.     

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.     

Anti-herpes simplex virus activity of alkaloids isolated from Stephania cepharantha

By screening water and MeOH extracts of 30 Chinese medicinal plants for their anti-herpes simplex virus (HSV)-1 activity, a MeOH extract of the root tubers of Stephania cepharantha HAYATA showed the most potent activity on the plaque reduction assay with an IC50 value of 18.0 microg/ml. Of 49 alkaloids isolated from the MeOH extract, 17 alkaloids were found to be active against HSV-1, including 13 bisbenzylisoquinoline, 1 protoberberine, 2 morphinane and 1 proaporphine alkaloids, while benzylisoquinoline and hasubanane alkaloids were inactive. Although N-methylcrotsparine was active against HSV-1, as well as HSV-1 thymidine kinase deficient (acyclovir resistant type, HSV-1 TK-) and HSV-2 (IC50 values of 8.3, 7.7 and 6.7 microg/ml, respectively), it was cytotoxic. FK-3000 was found to be the most active against HSV-1, HSV-1 TK- and HSV-2 (IC50 values of 7.8, 9.9 and 8.7 microg/ml) with in vitro therapeutic indices of 90, 71 and 81, respectively. FK-3000 was found to be a promising candidate as an anti-HSV agent against HSV-1, acyclovir (ACV) resistant-type HSV-1 and HSV-2.     

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.     

二羟丙氧甲基乌嘌呤抗单纯疱疹病毒Ⅰ型的实验研究

二羟丙氧甲基鸟嘌岭(DHPG),在组织培养中对单纯疱疹病毒Ⅰ型(HSV-1)KOS株抑制50%空斑形成的浓度(IC_(50))为0.1μg/ml。DHPG与环胞苷联合应用的抗HSV活性呈协同作用;DHPG分别与无环鸟苷、酞丁安合用有相加作用。0.1和0.05%DHPG溶液滴眼对家兔实验性浅层单纯疱疹病毒角膜炎有显著治疗作用,浓度降低至0.025和0.0l%无明显疗效。对实质层型单疱角膜炎,0.1%DHPG溶液滴眼治疗有效。     

Antiviral activity of seven iridoids, three saikosaponins and one phenylpropanoid glycoside extracted from Bupleurum rigidum and Scrophularia scorodonia

As part of our screening of antiviral agents from medicinal plants, 11 compounds from plant origin (Bupleurum rigidum and Scrophularia scorodonia), three saikosaponins, seven iridoids and one phenylpropanoid glycoside were tested in vitro against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. Five of these compounds showed antiviral activity against VSV. The percentages of cellular viability at the non-toxic limit concentrations of the active compounds were: verbascoside 53.6 % at 500 microg/ml, 8-acetylharpagide 32.1 % at 500 microg/ml, harpagoside 43.3 % at 450 microg/ml, scorodioside 47.8 % at 500 microg/ml and buddlejasaponin IV 56.9 % at 25 microg/ml. Although none of the saikosaponins were active against HSV-1, the iridoid scorodioside showed moderate in vitro anti-HSV-1 activity (30.6 % at 500 microg/ml). However, none of the compounds tested in this survey had any effect against poliovirus.     

Antiviral activity of Plantago major extracts and related compounds in vitro

Plantago major L., a popular traditional Chinese medicine, has long been used for treating various diseases varying from cold to viral hepatitis. The aim of present study was to examine the antiviral activity of aqueous extract and pure compounds of P. major. Studies were conducted on a series of viruses, namely herpesviruses (HSV-1, HSV-2) and adenoviruses (ADV-3, ADV-8, ADV-11). The antiviral activity of EC50 was defined as the concentration achieved 50% cyto-protection against virus infection and the selectivity index (SI) was determined by the ratio of CC50 (concentration of 50% cellular cytotoxicity) to EC50. Results showed that aqueous extract of P. major possessed only a slight anti-herpes virus activity. In contrast, certain pure compounds belonging to the five different classes of chemicals found in extracts of this plant exhibited potent antiviral activity. Among them, caffeic acid exhibited the strongest activity against HSV-1 (EC50=15.3 microg/ml, SI=671), HSV-2 (EC50=87.3 microg/ml, SI=118) and ADV-3 (EC50=14.2 microg/ml, SI=727), whereas chlorogenic acid possessed the strongest anti-ADV-11 (EC50=13.3 microg/ml, SI=301) activity. The present study concludes that pure compounds of P. major, which possess antiviral activities are mainly derived from the phenolic compounds, especially caffeic acid. Its mode of action against HSV-2 and ADV-3 was found to be at multiplication stages (postinfection of HSV-1: 0-12 h; ADV-3: 0-2 h), and with SI values greater than 400, suggesting the potential use of this compound for treatment of the infection by these two viruses.     

Cytotoxicities and anti-herpes simplex virus activities of diterpenes isolated from Euphorbia species

The cytotoxicities of nine diterpene polyesters obtained from Euphorbia species were assayed by measuring their effects on the growth of Vero cells. Their antiviral effects on the multiplication of Herpes simplex virus type 2 (HSV-2) were studied by using the virus yield reduction method in cell cultures. With the exception of the strongly cytotoxic 2alpha,5alpha,14beta-triacetoxy-3beta-benzoyloxy-8alpha,15beta-dihydroxy-7beta-isobutanoyloxy-9alpha-nicotinoyloxyjatropha-6(17),11E-diene (CC(50) 3.5 microg/ml), all the tested diterpenes exhibited a pronounced or moderate anti-herpes virus effect (IC(50) values between 2.5 and 8.3 microg/ml). The observed HSV-2 inhibitory activities were not associated with virucidal effects.     

Suppression of proliferation of poliovirus and porcine parvovirus by novel phenoxazines, 2-amino-4,4 alpha-dihydro-4 alpha-7-dimethyl-3H-phenoxazine-3-one and 3-amino-1,4 alpha-dihydro-4 alpha-8-dimethyl-2H-phenoxazine-2-one

The present study aimed at investigating the antiviral effects of 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2) on 6 representative viruses: poliovirus, porcine parvovirus, simian virus 40 (SV-40), herpes simplex virus-1 (HSV-1), Sindbis virus, and vesicular stomatitis virus (VSV). Phx-1 and Phx-2 suppressed the proliferation of poliovirus in Vero cells and that of porcine parvovirus in ESK cells at concentrations between 0.25 microg/ml and 2 microg/ml, when the cells were treated with Phx-1 and Phx-2 for 1 h and then inoculated with these viruses. The proliferation of the other viruses, SV-40, HSV-1, Sindbis virus, and VSV, in the host cells was not influenced by Phx-1 or Phx-2 at concentrations less than 20 microg/ml. The results suggest that Phx-1 and Phx-2 may be useful to prevent the proliferation of poliovirus and porcine parvovirus infection and may contribute to developing new antiviral drugs in future.     

Rapid determination of antiviral drug susceptibility of herpes simplex virus types 1 and 2 by real-time PCR

An antiviral drug susceptibility assay of herpes simplex virus (HSV) was developed using real-time PCR quantification of intracellular viral DNA load. The number of HSV DNA copies within Vero cells after 24 h infection was strongly correlated with the number of plaques obtained after 72 h infection. Antiviral drug susceptibility of HSV was determined after virus growth for 24h by measuring the reduction of intracellular HSV DNA in the presence of increasing concentrations of either acyclovir (ACV) or foscarnet (PFA). This assay required neither preliminary titration of infectious stock nor follow-up of cytopathic effect. The 50% inhibitory concentrations (IC50s) obtained with 27 isolates of HSV types 1 and 2 by using this test were significantly correlated with those obtained in parallel with plaque reduction assay taken as the reference method (r=0.91, p<0.0001 and r=0.51, p=0.009 for ACV and PFA, respectively). The threshold real-time PCR IC50s for ACV and PFA resistance did not differ according to HSV type and were determined to be 1.0 and 100 microM, respectively. The real-time PCR susceptibility assay reported here is rapid, reproducible, applicable for HSV-1 as well as HSV-2, and suitable for automation.     

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.     

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.     

Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (alpha-IFN). Using the colorimetric method, HSV isolates had ED50s ranging from 0.03 +/- 0.02 micrograms/ml to 0.164 +/- 0.03 micrograms/ml for ACV and 6.3 +/- 5.2 IU/ml to 55.0 +/- 11.4 IU/ml for alpha-IFN. With the DNA hybridization method, ED50s ranged from 0.033 +/- 0.012 micrograms/ml to 0.190 +/- 0.031 micrograms/ml for ACV and 8.5 +/- 5.0 IU/ml to 43.5 +/- 6.0 IU/ml for alpha-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (greater than 50.0 micrograms/ml). The values obtained by the two methods showed good correlation (r = 0.724, P = 0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.     

Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication.

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC50) of 28-DAS was 1.46 microg/ml without cytotoxicity at 400 microg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.     

Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase

Feline herpesvirus-1 (FHV-1) is the causative agent of a severe ocular disease in cats for which a safe potent antiviral chemotherapeutic agent is highly demanded. The sensitivity of FHV-1 to inhibition by three anti-herpetic nucleoside analogues [acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV)] was tested by means of yield reduction assay. ACV showed very poor ability to inhibit FHV-1 replication. At low multiplicity of infection (MOI), both PCV and CDV were nearly equally effective with IC50 values ranging between 6 and 8 microg/ml. However, when the MOI was raised to 3PFU/cell, the activity of CDV was markedly reduced (IC50 25 microg/ml), while that of PCV remained relatively low (IC50 10 microg/ml). Although FHV-1 is normally insensitive to ACV, it exhibited >1000-fold increase in sensitivity when the thymidine kinase (TK) encoded by herpes simplex virus-1 (HSV-1) was supplied in trans. Furthermore, three PCV-resistant FHV-1 variants selected in vitro were shown to carry mutations in the TK gene. Taken together, these data provided direct evidence that PCV is a potent selective inhibitor of FHV-1 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues.     

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.