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.     

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.     

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.     

Inhibition of herpes simplex virus DNA replication by ara-tubercidin

Preliminary studies of the biochemical basis for the antiviral activity of the pyrrolo[2,3-d]pyrimidine nucleoside ara-tubercidin were conducted. Herpes simplex virus DNA synthesis was 3-fold more sensitive to inhibition by ara-tubercidin than was cellular DNA synthesis. Partially purified herpes DNA polymerases were more sensitive to inhibition by ara-tubercidin 5'-triphosphate than were cellular polymerases alpha and beta. Inhibition of viral DNA polymerase was competitive with dATP and noncompetitive with dTTP. The results suggest that the viral DNA polymerase plays a significant role in the antiviral activity of ara-tubercidin.     

Inhibition of herpes simplex virus replication by Flos verbasci infusion

The preliminary phytochemical investigations have revealed the presence of flavonoids, iridoids, phenolic acids, saponins, amino acids, free sugars, and mucilages in the lyophilized infusion obtained from flowers of Verbascum thapsiforme Schrad. (FVI). Antiviral activity of the FVI on Herpes simplex type 1 virus (HSV-1) was studied in vitro by the yield reduction test. Decrease in the virus titer amounted to about 2.5 log at the non-toxic concentrations of FVI. The inhibitory effect of FVI on HSV studied by plaque reduction test in Vero cells showed that 50% inhibition of virus plaques occurred at 190 micrograms/ml. The virucidal effect of FVI on HSV was also shown.     

Notoginsenoside ST-4 inhibits virus penetration of herpes simplex virus in vitro

Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in raw and treated forms for a long time, led to the isolation of a new dammarane-type saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3β,12β,20(S)-tri-hydroxydammar-24-ene-3-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 2)-β-d-glu-copyranoside, based on the detailed analyses of the 1D and 2D NMR spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50% effective concentration (EC₅₀) values, determined by plaque reduction assay, were 16.47 ± 0.67 and 19.44 ± 1.16 μM for HSV-1 and HSV-2, respectively, whereas the 50% cytotoxic concentration (CC₅₀) determined by the XTT test on Vero cells was 510.64 ± 4.56 μM. As analyzed by attachment assay and penetration assay based on plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due to the penetration inhibition effects, which was confirmed by fluorescence microscopy observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore, notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.     

Notoginsenoside ST-4 inhibits virus penetration of herpes simplex virus in vitro

Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in raw and treated forms for a long time, led to the isolation of a new dammarane-type saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3β,12β,20(S)-tri-hydroxydammar-24-ene-3-O-β-d-xylopyranosyl-(1?→?2)-β-d-glucopyranosyl-(1?→?2)-β-d-glu-copyranoside, based on the detailed analyses of the 1D and 2D NMR spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50% effective concentration (EC(50)) values, determined by plaque reduction assay, were 16.47?±?0.67 and 19.44?±?1.16?μM for HSV-1 and HSV-2, respectively, whereas the 50% cytotoxic concentration (CC(50)) determined by the XTT test on Vero cells was 510.64?±?4.56?μM. As analyzed by attachment assay and penetration assay based on plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due to the penetration inhibition effects, which was confirmed by fluorescence microscopy observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore, notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.     

Effect of interferon on herpes simplex virus replication in murine macrophage-like cell lines

The effect of interferon on the replication of herpes simplex virus types 1 and 2 was studied in two murine macrophage-like cell lines which differ in their ability to synthesize interferon. Higher titers of herpes simplex virus type 1 occurred in PU5-1.8 cell cultures where interferon was not produced than in J774A.1 cell cultures where low amounts of interferon were produced. Herpes simplex virus type 2 replicated poorly in both types of cell cultures. Interferon synthesis was induced in J774A.1 cell cultures but not in PU5-1.8 cell cultures. Exogenously added interferon was shown to inhibit virus replication, however the restrictiveness of these cells to HSV replication was not relieved by treating cell cultures with anti-interferon serum. These results show that factors other than induced interferon regulate the replication of herpes simplex viruses in these cells and suggest that induced interferon synthesis does not affect herpes simplex virus replication in macrophages.     

Evaluation of some pyrazoloquinolines as inhibitors of herpes simplex virus type 1 replication

Three structurally related aminopyrazoloquinoline derivatives were evaluated for their antiviral activity against Herpes Simplex virus type 1. These compounds were examined for their in vitro antiviral activity by two different bioassays, namely; crystal violet staining and tetrazolium dye (MTS) measurement. The antiviral role of these compounds was confirmed by enumerating the infectious particles with plaque assay. The acute toxicity values of the biologically active compounds were determined prior to their screening as antiviral agents.     

Effect of chlordane on influenza type A virus and herpes simplex type 1 virus replication in vitro

The effect of chlordane on the susceptibility of Madin-Darby canine kidney cells and African Green monkey kidney cells to infection with influenza type A/PR/8/34 (HON1) virus and herpes simplex type 1 virus was determined. Exposure of both cell lines to various concentrations of chlordane for 24 h at 37 degrees C (acute exposure) effected a marked reduction in the efficiency of influenza type A virus infection, except at a dose of 0.025 ppm. Acute exposure of the monkey cells did not alter their susceptibility to herpes simplex virus infection. Viral adsorption studies at 4 and 37 degrees C revealed a marked reduction in the attachment of influenza type A virus to both cell lines following acute exposure to 10 ppm chlordane. Viral inactivation studies carried out at 4 and 37 degrees C failed to reveal differences in the level of influenza type A virus inactivation in the presence or absence of chlordane. Madin-Darby canine kidney cells exposed to 10 ppm chlordane for 60 d (chronic exposure) manifested a decrease in the efficiency of influenza type A virus infection, whereas cells chronically exposed to 0.025 ppm chlordane manifested an increase in the efficiency of influenza type A virus infection relative to mock-treated control cells. When chronically exposed cells were passaged six times in the absence of chlordane, these effects were reversed. Viral adsorption studies carried out at 4 and 37 degrees C on cells chronically exposed to 10 ppm chlordane revealed a decrease in the adsorption of influenza type A virus. Quantitation of the levels of cell-surface sialic acid, the essential terminal sugar on the receptor for influenza type A virus, indicated that the reduced adsorption of influenza type A virus to Madin-Darby canine kidney cells was not due to a loss of cell-surface sialic acid. Our findings indicate that chlordane alters the susceptibility of cells to infection with influenza type A virus but not to herpes simplex type 1 virus.     

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.     

Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo

Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.     

Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.     

L-thymidine is phosphorylated by herpes simplex virus type 1 thymidine kinase and inhibits viral growth.

We have demonstrated that herpes simplex 1 (HSV1) thymidine kinase (TK) shows no stereospecificity for D- and L-beta-nucleosides. In vitro, L enantiomers are not recognized by human TK, but function as specific substrates for the viral enzyme in the order: L-thymidine (L-T) > 2'-deoxy-L-guanosine (L-dG) > 2'-deoxy-L-uridine (L-dU) > 2'-deoxy-L-cytidine (L-dC) > 2'-deoxy- L-adenosine (L-dA). HSV1 TK phosphorylates both thymidine enantiomers to their corresponding monophosphates with identical efficiency and the Ki of L-T (2 microM) is almost identical to the Km for the natural substrate D-T (2.8 microM). The L enantiomer reduces the incorporation of exogenous [3H]T into cellular DNA in HeLa TK-/HSV1 TK+ but not in wild-type HeLa cells, without affecting RNA, protein synthesis, cell growth, and viability. L-T markedly reduces HSV1 multiplication in HeLa cells. Our observations could lead to the development of a novel class of antiviral drugs characterized by low toxicity.     

U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication

The level of cholesterol in host cells has been shown to affect viral infection. However, it is still not understood why this level of regulation is important for successful infection. We have shown in this study that dengue virus infection was affected when the cholesterol intake in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. The antiviral effect was found to result from two events: retarded viral trafficking in the cholesterol-loaded late endosomes/lysosomes and suppressed de novo sterol biosynthesis in treated infected cells. We also observed an additive antiviral effect of U18666A with C75, a fatty acid synthase inhibitor, suggesting dengue virus relies on both the host cholesterol and fatty acid biosynthesis for successful replication.     

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 micromolar concentrations of delta-9-tetrahydrocannabinol on herpes simplex virus type 2 replication in vitro

The effect of micromolar concentrations of delta-9-tetrahydrocannabinol (delta-9-THC) on the in vitro replication and biosynthesis of herpes simplex virus type 2 (HSV2) was determined. A 100-fold increase in extracellular virus was recorded for infected Vero cells pretreated with 10(-6) M or 10(-5) M drug when compared to infected vehicle-treated controls. However, no significant differences were observed in the production of total infectious virus for any of the vehicle or drug-treated cultures. Immunofluorescence of virus-infected cells revealed that delta-9-THC did not alter the intracellular compartmentalization of virus-specified proteins. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of isotopically labeled, cell-associated virus-specified proteins revealed that delta-9-THC had no major effect on the production of early nonstructural proteins but decreased the synthesis of late structural proteins. Scanning electron microscopy and light microscopy revealed blebs on the surface and macrovacuoles in the cytoplasm of both infected and uninfected cells treated with drug. These results suggest that delta-9-THC at micromolar concentrations selectively targets the host cell with the consequence of perturbation of cellular membranes. The alteration of cellular membranes may account for the enhanced virus release and for the decreased expression of virus-specified, cell-associated late structural proteins.