Resveratrol inhibition of herpes simplex virus replication.

Resveratrol, a phytoalexin, was found to inhibit herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) replication in a dose-dependent, reversible manner. The observed reduction in virus yield was not caused by the direct inactivation of HSV by resveratrol nor inhibition of virus attachment to the cell. The chemical did, however, target an early event in the virus replication cycle since it was most effective when added within 1 h of cell infection, less effective if addition was delayed until 6 h post-infection and not effective if added 9 h post-infection. Resveratrol was also found to delay the cell cycle at S-G2-M interphase, inhibit reactivation of virus from latently-infected neurons and reduce the amount of ICP-4, a major immediate early viral regulatory protein, that is produced when compared to controls. These results suggest that a critical early event in the viral replication cycle, that has a compensatory cellular counterpart, is being adversely affected.     

Virucidal activity of a beta-triketone-rich essential oil of Leptospermum scoparium (manuka oil) against HSV-1 and HSV-2 in cell culture

The inhibitory activity of manuka oil against Herpes simplex virus type 1 (HSV-1) and Herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells (monkey kidney cells) using a plaque reduction assay. In order to determine the mode of antiviral action of the essential oil, manuka oil was added at different times to the cells or viruses during the infection cycle. Both HSV types were significantly inhibited when the viruses were pretreated with manuka oil 1 h prior to cell infection. At non-cytotoxic concentrations of the essential oil, plaque formation was significantly reduced by 99.5 % and 98.9 % for HSV-1 and HSV-2, respectively. The 50 % inhibitory concentration (IC (50)) of manuka oil for virus plaque formation was determined at 0.0001 % v/v ( = 0.96 microg/mL) and 0.00006 % v/v ( = 0.58 microg/mL) for HSV-1 and HSV-2, respectively. On the other hand, pretreatment of host cells with the essential oil before viral infection did not affect plaque formation. After virus penetration into the host cells only replication of HSV-1 particle was significantly inhibited to about 41 % by manuka oil. Flavesone and leptospermone, two characteristic ss-triketones of manuka oil, inhibited the virulence of HSV-1 in the same manner as the essential oil itself. When added at non-cytotoxic concentrations to the virus 1 h prior to cell infection, plaque formation was reduced by 99.1 % and 79.7 % for flavesone and leptospermone, respectively.     

Broad-spectrum antiviral activity of the acyclic guanosine phosphonate (R,S)-HPMPG

(R,S)-9-(3-hydroxy-2-phosphonomethoxypropyl)guanine [(R,S)-HPMPG] exhibits broad spectrum antiviral activity with an ED50 of less than 1 microM against herpes simplex virus (HSV) types 1 and 2, varicella zoster virus, human cytomegalovirus (HCMV) and vaccinia in plaque reduction assays. Wild type HSV-2 and its thymidine kinase deficient variant are equally sensitive to (R,S)-HPMPG. (R,S)-HPMPG is 100-fold more potent than acyclovir (ED50 = 0.45 microM vs. 44 microM, respectively) against HCMV in cell culture, and 10-fold more active than acyclovir in extending survival time in mice intraperitoneally infected with 70 LD50 HSV-1. However, (R,S)-HPMPG is toxic when administered repeatedly at 44 mg/kg/day in uninfected adult mice. The diphosphoryl derivative of HPMPG was enzymatically synthesized and is a competitive inhibitor of HSV-1 DNA polymerase relative to dGTP (K1 = 0.03 microM). HPMPG-PP is 70-fold less active at inhibiting HeLa DNA polymerase alpha than HSV-1 DNA polymerase. At concentrations between 0.3 and 1.5 microM (R,S)-HPMPG inhibited HSV-1 DNA replication greater than or equal to 50% in infected cells as measured by nucleic acid hybridization. Consistent with inhibition of viral DNA synthesis, 6 to 30 microM (R,S)-HPMPG reduces late viral polypeptide synthesis in HSV-1 infected cells. These data indicate that (R,S)-HPMPG is a thymidine kinase independent broad spectrum antiviral drug which is capable of inhibiting viral DNA polymerase.     

The alkaloid 4-methylaaptamine isolated from the sponge Aaptos aaptos impairs Herpes simplex virus type 1 penetration and immediate-early protein synthesis

We describe in this paper that the alkaloid 4-methylaaptamine, isolated from the marine sponge Aaptos aaptos, inhibited HSV-1 infection. We initially observed that 4-methylaaptamine inhibited HSV-1 replication in Vero cells in a dose-dependent manner with an EC50 value of 2.4 microM. Moreover, the concentration required to inhibit HSV-1 replication was not cytotoxic, since the CC50 value of 4-methylaaptamine was equal to 72 microM. Next, we found that 4-methylaaptamine sustained antiherpetic activity even when added to HSV-1-infected Vero cells at 4 h after infection, suggesting that this compound inhibits initial events during HSV-1 replication. We observed that 4-methylaaptamine impaired HSV-1 penetration without affecting viral adsorption. In addition, the tested compound could inhibit, in an MOI-dependent manner, the expression of an HSV-1 immediate-early protein, ICP27, thus preventing the inhibition of macromolecular synthesis induced by this virus. Our results warrant further investigation on the pharmacokinetics of 4-methylaaptamine and propose that this alkaloid could be considered as a potential compound for HSV-1 therapy.     

Yucca leaf protein (YLP) stops the protein synthesis in HSV-infected cells and inhibits virus replication.

Yucca leaf protein (YLP), an inhibitor of tobacco mosaic virus isolated from the leaves of Yucca recurvifolia Salisb., exhibited potent activity against herpes simplex virus type 1 (HSV-1) with no cytotoxicity below 300 micrograms/ml. The inhibitory dose was varied with the time of addition; 50% effective concentrations (ED50) of YLP were 3, 19 and 95 micrograms/ml when YLP exposure was begun 3 h before virus infection, 0 h and 3 h after infection, respectively. This protein also inhibited the multiplication of herpes simplex virus type 2 and human cytomegalovirus. YLP has been shown to have a weak virucidal activity at higher concentrations. Analysis of early events following infection showed that YLP affected viral penetration in HeLa cells but did not interfere with adsorption to the cells. YLP was found to exert strong inhibition of protein synthesis in virus-infected cells but not in uninfected cells. This selective effect can be considered to attribute mainly to the antiviral activity of YLP.     

Acetylenic nucleosides. 4. 1-beta-D-arabinofuranosyl-5-ethynylcytosine. Improved synthesis and evaluation of biochemical and antiviral properties

5-Ethynyl-1-beta-D-arabinofuranosylcytosine (EAC) was prepared from 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)cytosine by iodination followed by coupling with (trimethylsilyl)acetylene and deblocking. At 50 microM, EAC was found to inhibit the in vitro replication of herpes simplex virus type 1 and type 2 by greater than 99%. EAC also showed activity against a strain of HSV-1 resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine which has an alteration of the virus-induced thymidine kinase (TK). At 100 microM, EAC did not inhibit the in vitro growth of leukemia L1210 and HeLa cells. EAC was resistant to the action of dCR-CR deaminase, its rate of deamination being approximately 2% that of dCR. The compound was a poor substrate for dCR kinase, but it was phosphorylated by HSV-1- and HSV-2-induced TKs at 50% and 30%, respectively, the rate of thymidine.     

Anti-herpesvirus activity of adenine arabinoside analogues in tissue culture and a genital infection of mice and guinea pigs

Four analogues of adenine arabinoside (ara-A) were compared for activity against herpes simplex virus (HSV) in tissue culture and in a genital infection of mice and guinea pigs. These analogues, 5'-monophosphate (ara-AMP), 5'-valerate ester (ara-AV), 2'3'-diacetate ester (ara-ADA), and 2',3',5'- triacetate ester (ara-ATA) have greater water and lipid solubility and resistance to deamination than ara-A. In mouse embryo fibroblast cells, similar viral inhibitory levels were noted with ara-A, AMP, and ara-Av, while ara-ADA and ara-ATA were 6-10 time less active. In mice infected intravaginally with HSV type 2 (HSV-2), intravaginal treatment with 10% concentrations of each of the compounds beginning 3 h after viral challenge, had no effect on infection rates, titers of virus in vaginal secretions, mortality rates or the mean day of death as compared with placebo-treated controls. In the HSV-2 genital infection of guinea pigs, treatment with 10% vaginal creams or placebo vehicle was initiated 6 or 24 h after viral inoculation. In animals treated at 6 h with ara-A, ara-AMP and ara-AV, there was complete inhibition of viral replication in the vaginal tract and development of external genital lesions. When treatment with these three drugs was delayed 24 h after infection, there was no effect on vaginal virus titers, but lesions severity was reduced by ara-A or ara-AMP therapy. Ara-ATA was ineffective whether begun at 6 or 24 h. The greater solubility in water and lipid as well as the resistance to deamination of ara-AMP and ara-AV did not appear to enhance their antiviral activity over that of ara-A. Additionally, ara-ADA and ara-ATA exhibited less activity both in tissue culture and in the experimental genital infections.     

Houttuynia cordata blocks HSV infection through inhibition of NF-κB activation

Houttuynia cordata Thunb. is a medicinal plant widely used in folk medicine in several Asian countries. It has been reported that a water extract of H. cordata exhibits activity against herpes simplex virus (HSV) and the virus of severe acute respiratory syndrome (SARS), although the mechanisms are not fully understood yet. Previous studies have demonstrated absolute requirement of NF-κB activation for efficient replication of HSV-1 and HSV-2 and inhibition of NF-κB activation has been shown to suppress HSV infection. Here we show that a hot water extract of H. cordata (HCWE) inhibits HSV-2 infection through inhibition of NF-κB activation. The IC₅₀ was estimated at 50 μg/ml of lyophilized HCWE powder. At 150 and 450 μg/ml, HCWE blocked infectious HSV-2 production by more than 3 and 4 logs, respectively. The inhibitory activity was concomitant with an inhibition of NF-κB activation by HSV-2 infection. Although activation of NF-κB and Erk MAPK has been implicated for HSV replication and growth, HCWE showed no effect on HSV-2-induced Erk activation. Furthermore, we show that treatment with quercetin, quercitrin or isoquercitrin, major water extractable flavonoids from H. cordata, significantly blocked HSV-2 infection. These results together demonstrated that H. cordata blocks HSV-2 infection through inhibition of NF-κB activation.     

Mechanism of the protective effect of heteropolyoxotungstate against herpes simplex virus type 2

The effects of heteropolyoxotungstate (K(7)[PTi(2)W(10)O(40)]. 6H(2)O; PM-19) on the replication of herpes simplex virus type 2 (HSV-2) were examined using a semiquantitative polymerase chain reaction of intracellular viral DNA established by us and also other methods. Vero cells were infected with HSV-2 strains: either the standard strain 169, or the acyclovir-resistant strain YS-4C-1. PM-19 was added at various stages during the replication of HSV-2. PM-19 strongly inhibited the synthesis of viral genomic DNA when it was added at the time of infection. The addition of PM-19 60-90 min after viral inoculation time-dependently decreased the antiviral activity and increased the relative yield of viral DNA, and the addition of PM-19 was completely ineffective at times later than 90 min. These results suggested that PM-19 inhibited viral penetration but did not affect the synthesis of viral DNA. Furthermore, PM-19 strongly inhibited a second round of infection.     

Inhibitory potential of Crotalus durissus terrificus venom on measles virus growth.

This paper presents the antiviral activity found in a snake with Crotalus durissus terrificus venom (Cdt), studied by use of microplate inhibition assay, using measles virus (MV). Cdt at concentrations below 100 microg/ml showed no cytotoxicity for Vero cells. This study shows the optimal conditions for cell treatment and infection. Two factors that affect virus binding and infection efficiency were studied: the use of an adsorption step, where infection volume was varied; and the concentration of fetal bovine serum (FBS). The adsorption step, with or without FBS, increased the bound virus percentage, whereas it increased bound virus at equilibrium only in FBS-free until 2.5% FBS. In contrast, the addition of 10% FBS decreased the bound virus percentage. The inhibition of MV replication in Vero cells was observed when Cdt was added either before or during cell infection with virus. Its inhibitory concentration against MV replication was 0.1 until 100 microg/ml, respectively. The anti-MV effect of the Cdt was gradually decreased when it was added before or during infection, and little inhibition was observed when Cdt was added 1 h after infection, suggesting that the MV infection was inhibited at the time of the initial events such as at the moment of adsorption and penetration of the viral cycle. In conclusion, Cdt contains anti-MV effects that may be of potential clinical interest.     

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1

The polyphenole-enriched acetone-water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC(50)) and cytotoxic concentration (CC(50)) were determined. R2 exibited an IC(50) of 0.8 μg/mL and a selectivity index (SI) (ratio of IC(50) to CC(50)) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure-activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1). R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥ 12.5 μg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD. Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.     

Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells

Partially purified extracts from leaves of Melia azedarach L. (MA) exert a broad range of antiviral effects on DNA and RNA viruses. The effect of MA on different stages of Sindbis virus replicative cycle in BHK cells was investigated. Under one-step growth conditions MA afforded a greater than 90% inhibition in virus yield if added to the cell cultures 2 h before or after infection, and when added 4 h after infection MA still caused a greater than 80% inhibition. Analysis of early events following Sindbis virus infection showed that MA did not affect viral adsorption to or penetration in BHK cell. In contrast, viral RNA and protein synthesis was almost totally inhibited in cells pretreated with MA 2 h before infection, while cellular macromolecular synthesis was similar in MA-treated and untreated cell cultures.     

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.     

Inhibition of herpes simplex virus types 1 and 2 replication in vitro by mercurithio analogs of deoxyuridine.

The in vitro antiviral activity of several 5-mercurithio analogs of 2'-deoxyuridine (dUrd) on the replication of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were examined. Of those compounds tested, the thioglycerol analog of 5-mercuri-2'-deoxyuridine (HgdUrd) was most effective in inhibiting the replication of HSV-1 in KB cells with a 50% inhibitory dose (ID50) of 0.001 micrograms/ml while the glutathione analog of HgdUrd was the most effective in inhibiting the replication of HSV-2 with a ID50 of 0.075 micrograms/ml. Conversely in HeLa TK- cells, the mercaptoguanosine analog of HgdUrd was the most effective compound in inhibiting virus replication with ID50S of 0.098 and 0.001 micrograms/ml for HSV-1 and HSV-2 respectively. These results suggest that these mercurithio analogs of dUrd are as effective as acyclovir in preventing the replication of these herpesviruses.     

Effect of resveratrol on herpes simplex virus vaginal infection in the mouse

Resveratrol (3,5,4'-trihydroxystilbene) is a natural component of certain foods, such as grapes, that, when topically applied, has been shown to limit HSV-1 lesion formation in the skin of mice [Antiviral Res. 61:19-26, 2004]. To determine if it is active on genital HSV infection, the vagina of mice were infected with HSV-2 or HSV-1 and treated with a cream formulation of resveratrol. Mice were evaluated daily for extravaginal disease and vaginal swabs were taken regularly and assayed for infectious virus. Initial studies demonstrated that 19% resveratrol cream administered intravaginally five times a day for 5 days beginning 1h after infection significantly reduced HSV-2 replication beginning on day 1 of infection and prevented extravaginal disease when compared to animals treated with placebo. When resveratrol was tested at a concentration of 6.25% and 12.5% administered five times a day, 6.25% limited virus replication only on day 1 and delayed development of extravaginal disease by 1 day. However, 12.5% resveratrol inhibited HSV-2 replication beginning on day 1 and abolished extravaginal disease. If the number of applications per day was reduced to three for 5 days, 12.5% resveratrol inhibited HSV-2 replication only on day 1, while 19% resveratrol inhibited it throughout the 9-day assay period. When the animals with three treatments per day were examined for extravaginal disease, it was found that 12.5% resveratrol was ineffective when compared to placebo, while animals treated with 19% resveratrol did not exhibit extravaginal disease. When treatment was delayed 6h, 12.5% resveratrol did not inhibit HSV-2 replication or extravaginal lesion formation, but 19% resveratrol did. When resveratrol was used to treat vaginal HSV-1 infection, it was found that 12.5% resveratrol did not limit replication or prevent extravaginal lesion formation. In contrast, 19% resveratrol did significantly limit vaginal HSV-1 replication and reduced extravaginal lesion formation, but the latter was not significant. Mortality rates in placebo-treated animals was 37%, 6.25% resveratrol-treated animals was 40%, 12.5% resveratrol-treated animals was 24%, and 19% resveratrol-treated animals was 3%. Collectively, these results demonstrate that resveratrol cream inhibits or reduces HSV replication in the vagina of mice and limits extravaginal disease.     

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.     

Inhibition of herpes simplex virus type 2 replication in vitro by 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A

The in vitro antiviral activity as well as the mechanism of action of a new antiviral agent, a kanamycin analogue, 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A (PTKA) against herpes simplex virus type 2 (HSV-2) was investigated. The drug showed excellent antiviral action with negligible cytotoxic effect on the culture cells. Based on plaque reduction assays the 50% inhibitory dose (ID50) of the drug was 1 microgram/ml, and at 20 micrograms/ml plaque formation was totally suppressed. The compound inhibited viral protein synthesis in infected cells without affecting RNA and DNA synthesis, when added to the cultures after virus adsorption. Moreover, pretreatment of the cells with PTKA before HSV-2 infection, increased the antiviral activity significantly. Dot-blot hybridization analysis revealed that the drug reduced the level of immediate early viral mRNA if applied before infection. There was no detectable action at the level of virus adsorption, penetration or uncoating. These results indicate that PTKA exerted its antiviral action at the early stage of viral replication as well as at the level of viral protein synthesis.