Antiherpetic mode of action of (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one in vitro

The replication of herpes simplex virus (HSV) type 1 in Vero cells is inhibited in the presence of (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b), a synthetic brassinosteroid derivative. Attempts to disclose the mode of action of 6b indicate that a late step of virus multiplication is affected. In the presence of 6b, HSV late protein synthesis was severely diminished and this inhibitory effect of 6b on HSV antigen expression was confirmed by immunofluorescence assays.     

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.     

Cell culture replication of herpes simplex virus and,or human cytomegalovirus is inhibited by 3,7-dialkoxylated, 1-hydroxyacridone derivatives

The synthetic acridone compound, 5-chloro-1,3-dihydroxyacridone inhibits herpes simplex virus (HSV) replication by inducing the formation of defective viral (B-type) capsids [Antiviral Res. 53 (2002) 113]. In this report, synthetic elaboration of the 1-hydroxyacridone scaffold coupled with antiviral testing led to the identification of 3,7-dimethoxy-1-hydroxy-acridone (2) as an inhibitor of low multiplicity human cytomegalovirus (HCMV) infection (ED(50) value of 1.4 microM (0.5 microg/ml); greater than 35-fold selectivity). Compound 2 was inactive against HSV replication and the efficacy as an anti-HCMV agent at higher viral loads was only apparent if host cells were replicated in the presence of the compound prior to infection. Interestingly, the 3,5-dimethoxy regioisomer inhibited cell replication (mean CC(50) 33 microM) and was inactive as a selective anti-herpes agent. A limited parallel synthesis and testing of ten 3,7-dialkoxylated compounds closely related to compound 2 led to the discovery of the 3-ethoxy-, 3-propoxy-, 3-isopropoxy- and 3-allyloxy-derivatives as dual inhibitors of both HSV and HCMV (selectivity of the 3-allyloxy analog was greater than 10- and 36-fold, respectively). The 3-benzyloxy-derivative was active (ED(50) value of 6.9 microM) against HCMV only. Moreover, the corresponding C-7 variable alkoxylated parallel series were either weakly active or inactive antiviral agents suggesting an apparent requirement for a C-7 methoxy substituent in the active structure. Exploratory mode of action studies showed that dual inhibitors were most active against a low multiplicity HSV infection and potent inhibition of viral release likely contributed to this. Furthermore, suppression of late viral protein synthesis by dual inhibitors did not correlate with anti-HSV activity. On the basis of the present findings, the 1-hydroxyacridone scaffold is further expanded as a useful template for the discovery of investigational anti-herpes agents. As a group, the active 3,7-dialkoxylated compounds likely have diverse mechanisms of action, consequently they are of potential medicinal interest.     

Antiviral activity of enterocin CRL35 against herpesviruses

Enterocin CRL35 is an antibacterial polypeptide of 3.5×10³ Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk⁺) and deficient (tk⁻) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 μg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.     

Tromantadine inhibits a late step in herpes simplex virus type 1 replication and syncytium formation

Addition of tromantadine after virus penetration inhibited HSV-1 induced syncytium formation and virus production in HEp-2 and VERO cells and acted additively with neutralizing antibody in blocking virus spread and cytopathology. Inhibition of syncytium formation in VERO cells infected with 0.01 pfu/cell of HSV-1 GC+ was observed at a concentration greater than 25 micrograms/ml. The extent of inhibition was dependent upon the multiplicity of infection and cell type. Tromantadine inhibited a late event in HSV-1 replication which appeared to be sensitive to cycloheximide. Reversal of the inhibitory effect of tromantadine on syncytium formation required new protein synthesis. HSV-1 gB, gC, and gD were synthesized in the presence of tromantadine and could be detected on the cell surface by immunofluorescence. Tromantadine most likely inhibits a cellular process that is required for syncytium formation, such as glycoprotein processing, which occurs after the synthesis of the fusion protein but before its expression on the cell surface.     

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.     

Inhibition of herpes simplex virus replication by anthracycline compounds

The replication of type 1 and type 2 strains of herpes simplex virus (HSV) was inhibited greater than 99.9% by low concentrations (0.1-0.2 microM) of anthracycline compounds. The degree of viral inhibition was dependent upon the host cell. N,N-dimethyl daunomycin (NDMD), a non-mutagenic compound, was more potent as an inhibitor of HSV synthesis than either daunomycin (DM) or adriamycin (AD). The depression of viral yield by DM or AD was attributable, in part, to a temperature-dependent direct effect on infectious virions. Tritium-labeled DM bound tightly to HSV particles. NDMD did not directly inactivate virions in spite of superior potency in reducing viral yields. All three anthracyclines could be added late in the infectious cycle (6-8 h p.i.) and retain effectiveness. Cesium chloride density gradient analysis verified that viral DNA synthesis was blocked by addition of all three anthracyclines early in the infectious cycle. The inhibition of HSV replication was not a simple consequence of the suppression of host DNA synthesis since treatment of cells with compounds for 24 h before infection did not reduce virus yields even though host DNA synthesis was inhibited by 90%. Further, the kinetics of inhibition of cellular DNA synthesis by anthracyclines was similar in HFF or Vero cells but the degree of inhibition of virus replication was markedly different. The data suggest that anthracyclines with substitutions on the sugar moiety may be useful anti-herpes agents.     

Antiviral agents alter ability of HSV-2 to disrupt gap junctional intercellular communication between mammalian cells in vitro

In cultured mammalian cells (Vero), different antiviral agents change to differing degrees the ability of HSV2 to down-regulate gap junctions, each agent having a specific effect. Measured by intracellular electrodes, control cell populations showed 49-51% coupling, uninfected populations treated with acyclovir or SDS averaged 43-51% coupling while populations infected with HSV2 had coupling reduced to 8%. The antiviral agent acyclovir (1 microg/ml), which suppresses viral replication, failed to prevent this down regulation (final coupling ratio of 11%). A plant extract (250 microg/ml) from Pilostigma thonningii offered slightly more protection (final coupling ratio of 22%), while sodium dodecyl sulfate (SDS) (50 microM) afforded nearly complete protection (final coupling ratio of 40%). With SDS there was an initial down regulation to only 16% coupling by 3 h post infection, followed by a recovery of intercellular communication to near control levels by 24 h. While SDS was originally believed to alter the viral coat and prevent entry into the cell, our data are in agreement with recent studies which indicate that SDS treated viruses can enter into host cells, but in a severely diminished condition. Our data also suggest that the gap junction antagonist is brought into the cells as part of the entering virus.     

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.     

Inhibition of herpes simplex virus type 2 (HSV-2) viral replication by the dominant negative mutant polypeptide of HSV-1 origin binding protein.

UL9-C535C, the trans-dominant negative mutant polypeptide of herpes simplex virus type 1 (HSV-1) UL9 origin binding protein, is a potent inhibitor of HSV-1 viral DNA replication. This study focused on testing whether HSV-1 UL9-C535C and a genetically engineered UL9-C535C-encoding HSV-1 recombinant virus CJ83193 could inhibit herpes simplex virus type 2 (HSV-2) infection. First, a stable cell line, R-C535C, expressing a high level of UL9-C535C in the presence of tetracycline and little or no UL9-C535C in the absence of tetracycline was established. The single step growth experiment showed that like HSV-1, the de novo synthesis of HSV-2 could be suppressed approximately 1000-fold by UL9-C535C expressed in R-C535C cells in the presence of tetracycline. Secondly, compared with cells singly infected with HSV-2, co-infection of Vero cells with HSV-2 and CJ83193 reduced the replication efficiency of HSV-2 in co-infected cells by 30-40 fold in a single-step growth assay, which coincided with marked reduction in viral late gene expression, but not the expression of viral immediate-early genes. Taken together, in view of our recent demonstration that CJ83193 can serve as an effective vaccine in preventing HSV-1 infection in mice, one can generate a CJ83193-like HSV-2 recombinant virus that could potentially function as a new therapeutic class of recombinant viral vaccine against HSV-2 infection.     

Bupleurum kaoi inhibits Coxsackie B virus type 1 infection of CCFS-1 cells by induction of type I interferons expression.

Coxsackie B virus type 1 (CVB1) infection is known to cause high morbidity and mortality in children, however, there is no effective drug for treating this disease. The present study aimed to examine the antiviral activity of Bupleurum kaoi (BK), a popular herbal drug for treating viral and bacterial infections, against CVB1 infection and its mechanisms of action. Our data showed that BK neutralized the CVB1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC50 and EC50 values around 12.38 microg/ml and 50.93 microg/ml, respectively. Its CC50 and SI values were 883.56 microg/ml and 17.34, respectively. These results suggest that BK possessed anti-CVB1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 microg/ml. The time-of-addition studies showed that BK (50, 100 and 200 microg/ml) added at various time of preinfection (-1 to -3 h), coinfection (0 h) and postinfection (1-3 h) could inhibit CVB1 infection. Interestingly, BK also showed an inhibition on viral replication through the induction of IFN-alpha/beta expression. In conclusion, BK possessed antiviral activity against CVB1 infection. It interfered the early stage of viral replication and viral replication after infection through the induction of type I interferon expression.     

Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture

In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5 μM BQR reduced virus progeny production by >90%, revealing an EC₅₀ (drug concentration required to inhibit 50% of virus replication) of 0.017 μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.     

Fluoroquinolones inhibit human polyomavirus BK (BKV) replication in primary human kidney cells

Reactivation of human polyomavirus BK (BKV) may cause polyomavirus-associated nephropathy or polyomavirus-associated hemorrhagic cystitis in renal- or bone marrow-transplant patients, respectively. Lack of treatment options has led to exploration of fluoroquinolones that inhibit topoisomerase II and IV in prokaryotes and possibly large T-antigen (LT-ag) helicase activity in polyomavirus. We characterized the effects of ofloxacin and levofloxacin on BKV replication in the natural host cells – primary human renal proximal tubular epithelial cells (RPTECs). Ofloxacin and levofloxacin inhibited BKV load in a dose-dependent manner yielding a ∼90% inhibition at 150 μg/ml. Ofloxacin at 150 μg/ml inhibited LT-ag mRNA and protein expression from 24 h post infection (hpi). BKV genome replication was 77% reduced at 48 hpi and a similar reduction was found in VP1 and agnoprotein expression. At 72 hpi, the reduction in genome replication and protein expression was less pronounced. A dose-dependent cytostatic effect was noted. In infected cells, 150 μg/ml ofloxacin led to a 26% and 6% inhibition of cellular DNA replication and total metabolic activity, respectively while 150 μg/ml levofloxacin affected this slightly more, particularly in uninfected cells. Cell counting and xCELLigence results revealed that cell numbers were not reduced. In conclusion, ofloxacin and levofloxacin inhibit but do not eradicate BKV replication in RPTECs. At a concentration of ofloxacin giving ∼90% inhibition in BKV load, no significant cytotoxicity was observed. This concentration can be achieved in urine and possibly in the kidneys. Our results support a mechanism involving inhibition of LT-ag expression or functions but also suggest inhibition of cellular enzymes.     

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.     

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.     

Inhibitory effect of 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H - imidazole on HCMV DNA replication and permissive infection.

We found that Human Cytomegalovirus (HCMV) infection of human fibroblasts resulted in a dramatic increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation. Recently, drug mediated inhibition of p38 has been demonstrated to exhibit anti-viral activity against HIV (Shapiro, L., Heidenreich, K.A., Meintzer, M.D. and Dinarello, C.A., 1998. Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro. Proc. Natl. Acad. Sci. USA. 95, 7422-7426). Therefore, we examined the effect of a specific p38 kinase inhibitor on HCMV infection. Inhibiting p38 activity in HCMV infected cells by treating cells with 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole; (FHPI), a p38 inhibitor drug, prevented permissive HCMV infection as measured by plaque assay. In the presence of FHPI, HCMV immediate early gene expression was slightly lower at early times of infection, but there was no inhibition of expression of the early gene UL-84, an HCMV protein essential for viral replication. However, FHPI inhibited HCMV DNA replication and late gene expression. The inhibitory effect of FHPI was reversible, as demonstrated by the induction of HCMV replication upon withdrawal of FHPI. Our data describes FHPI as a novel anti-HCMV compound that inhibits synthesis/activation of cellular and/or viral factors required for initiation of HCMV DNA replication.     

Xiao chai hu tang inhibits CVB1 virus infection of CCFS-1 cells through the induction of Type I interferon expression

Coxsackie B virus type 1 (CVB1) infection is known to cause high morbidity and mortality in children, however, there is no effective drug for treating this disease. The present study aimed to examine the antiviral activity of xiao chai hu tang (XCHT), a popular herbal drug for treating viral and bacterial infections, against CVB1 infection and its mechanisms of action. Our data showed that XCHT neutralized the CVB1-induced cytopathic effect in human neonatal foreskin fibroblast cell line (CCFS-1/KMC), with IC50 (virus-induced cytopathic effect by 50%) and EC50 (concentration of 50% effectiveness) values around 12.39 and 50.93 microg/ml, respectively. Its CC50 (concentration of 50% cellular cytotoxicity) and SI (selective index) values were 945.75 microg/ml and 18.92, respectively. These results suggest that XCHT possessed anti-CVB1 activity, and showed no effect on CCFS-1 cell viability and growth at concentration 250 microg/ml. The time-of-addition studies showed that XCHT (50, 100 and 200 microg/ml) added at various time of preinfection (-1 to -3 h), coinfection (0 h) and postinfection (1 approximately 3 h) could inhibit CVB1 infection. Interestingly, XCHT also showed an inhibition on viral replication through the induction of IFN-alpha/beta expression. In conclusion, XCHT possessed antiviral activity against CVB1 infection. It interfered the early stage of viral replication (prophylactic effect) and viral replication after infection (therapeutic effect) through the induction of Type I interferon expression.     

Antiviral property and mode of action of a sulphated polysaccharide from Sargassum patens against herpes simplex virus type 2

A sulphated polysaccharide (SP2) was isolated from the brown alga Sargassum patens. SP2 inhibited the replication of herpes simplex virus type 2 (HSV-2) dose-dependently by 38.5-96.1% of the control level, after incubations with 0.78-12.5 microg/ml of the polysaccharide. SP2 exhibited extracellular virucidal activity only in high concentrations (>/=12.5 microg/ml) but significantly inhibited the virus attachment to its host cells by 45.1%, at concentration as low as 1 microg/ml. All the results from this study suggested that the antiviral mode of action of SP2 could be ascribed to the inhibition of virus adsorption, which is different from that of the current drug of choice acyclovir.