Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Human natural killer cells limit replication of herpes simplex virus type 1 in vitro

Studies were undertaken to determine whether natural killer (NK) cells could inhibit the replication of herpes simplex virus type 1 (HSV-1) in culture. In the absence of effector cells, HSV-1 was found to replicate in fibroblasts with up to a 100-fold increase in virus titer from 4 to 16 hr after incubation at 37 degrees C. Human peripheral blood mononuclear cells were found to limit virus replication in a dose-dependent manner, with the greatest inhibition being observed at the highest concentration evaluated: i.e., an effector:target ratio of 800:1. The antiviral effect was not observed when nonactivated or virus-activated mononuclear cells were added to the virus preparations at the end (instead of the beginning) of the assay period, indicating that the observed effect was not due to a nonspecific toxicity of soluble factors released from freeze-thawed effectors. Neither was inhibition of HSV-1 replication due to the generation of interferon (IFN) during the NK assay, because the addition of anti-IFN did not abrogate the antiviral effect. Thus, the inhibition of viral replication was most likely due to a cytotoxic effector rather than to release of soluble factors. The effector cells responsible for limiting HSV-1 replication were shown to be NK cells by a number of criteria. Mononuclear cells from both HSV-1 seropositive and seronegative donors limited virus replication; their activity could be boosted by pretreatment of effector cells with IFN; the effector cells which limited virus replication were found in Percoll gradient fractions enriched for large granular lymphocytes; and the effector cells shared the cell surface phenotype of NK cells--they were enriched in populations depleted of T cells by panning with Leu-4 and were depleted of activity by treatment with the anti-NK antibody Leu-11b plus complement. We conclude that human NK cells are capable of recognizing and lysing HSV-1-infected target cells before infectious virus progeny are generated. These results suggest that NK cells, acting early in the course of an infection, might serve to limit HSV-1 replication and therefore reduce the virus load in the host before the development of the adaptive immune response and clearance of the infection.     

Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections

Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.     

Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea

An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

Cell type-dependent requirement of autophagy in HSV-1 antiviral defense

Type I interferons (IFNs) are induced during most viral infections and are considered to be the primary and universal means of innate viral control. However, several other innate mechanisms, including autophagy, have recently been shown to play an important role in antiviral defense. In our recent study, we utilized a herpes simplex virus 1 (HSV-1) infection model to investigate the relationship between cell type and innate antiviral immune mechanisms. Our study demonstrates that dorsal root ganglion (DRG) neurons undergo an innate antiviral response to HSV-1 that differs from the antiviral program induced in mitotic cells in three distinct ways. First, DRG neurons produce less type I IFN and undergo a less effective IFN antiviral program vs. mitotic cells in response to HSV-1 infection. Second, the type I IFN program initiated in DRG neurons induces less cell death than in mitotic cells. Third, in the absence of a robust type I IFN response, DRG neurons, but not mitotic cells, repy on autophagy in HSV-1 defense. Our findings reveal a cell type-specific requirement for autophagy in defense against HSV-1, and offer insight into the cell-appropriate antiviral defense mechanism employed by neurons.     

In vitro inhibition of herpes simplex virus type 1 replication by Mentha suaveolens essential oil and its main component piperitenone oxide

Several essential oils exert in vitro activity against bacteria and viruses and, among these latter, herpes simplex virus type 1 (HSV-1) is known to develop resistance to commonly used antiviral agents. Thus, the effects of the essential oil derived from Mentha suaveolens (EOMS) and its active principle piperitenone oxide (PEO) were tested in in vitro experimental model of infection with HSV-1. The 50% inhibitory concentration (IC₅₀) was determined at 5.1 μg/ml and 1.4 μg/ml for EOMS and PEO, respectively. Australian tea tree oil (TTO) was used as control, revealing an IC₅₀ of 13.2 μg/ml. Moreover, a synergistic action against HSV-1 was observed when each oil was added in combination with acyclovir. In order to find out the mechanism of action, EOMS, PEO and TTO were added to the cells at different times during the virus life-cycle. Results obtained by yield reduction assay indicated that the antiviral activity of both compounds was principally due to an effect after viral adsorption. Indeed, no reduction of virus yield was observed when cells were treated during viral adsorption or pre-treated before viral infection. In particular, PEO exerted a strong inhibitory effect by interfering with a late step of HSV-1 life-cycle. HSV-1 infection is known to induce a pro-oxidative state with depletion of the main intracellular antioxidant glutathione and this redox change in the cell is important for viral replication. Interestingly, the treatment with PEO corrected this deficit, thus suggesting that the compound could interfere with some redox-sensitive cellular pathways exploited for viral replication. Overall our data suggest that both EOMS and PEO could be considered good candidates for novel anti-HSV-1 strategies, and need further exploration to better characterize the targets underlying their inhibition.     

Specific phosphorylation of 5-ethyl-2'-deoxyuridine by herpes simplex virus-infected cells and incorporation into viral DNA

5-Ethyl-2'-deoxyuridine (EDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), which is currently being pursued for the topical treatment of HSV-1 and HSV-2 infections in humans. Using [4-14C]EDU as the radiolabeled analogue of EDU, it was ascertained that, at antivirally active doses, EDU is phosphorylated to a much greater extent by HSV-infected Vero cells than by mock-infected cells. Within the HSV-1-infected cells, EDU was incorporated to a much greater extent into viral DNA than cellular DNA. Using varying doses of EDU, a close correlation was found between the incorporation of EDU into viral DNA, the inhibition of viral DNA synthesis, and the inhibition of virus yield. It is postulated that the selectivity of EDU as an antiviral agent depends on both its preferential phosphorylation by the virus-infected cell and its preferential incorporation into viral DNA. The latter than results in a suppression of viral DNA synthesis and, hence, shutoff of viral progeny formation.     

The inhibitory effect of essential oils on herpes simplex virus type-1 replication in vitro

The antiviral effect of 12 essential oils on herpes simplex virus type-1 (HSV-1) replication was examined in vitro. The replication ability of HSV-1 was suppressed by incubation of HSV-1 with 1% essential oils at 4 C for 24 hr. Especially, lemongrass completely inhibited the viral replication even at a concentration of 0.1%, and its antiviral activity was dependent on the concentrations of the essential oil. When Vero cells were treated with the essential oil before or after viral adsorption, no antiviral activity was found, which suggests that the antiviral activity of essential oils including lemongrass may be due to the direct interaction with virions.     

Anti-herpesviral property and mode of action of a polysaccharide from brown seaweed (<Emphasis Type="Italic">Hydroclathrus clathratus</Emphasis>)

A sulfated polysaccharide, designated HC-b1, was isolated from the brown seaweed Hydroclathrus clathratus. It was found to be a strong inhibitor of herpes simplex virus type 1 (HSV-1), including acyclovir-resistant strain and clinical strain. HC-b1 inhibited the plaque formation of HSV-1 in a dose-dependent manner. It could protect Vero cells from infection by HSV-1 if the cells were incubated with HC-b1 before exposure to the virus. It also had inactivating effect against HSV-1 since the pretreatment of the virus with HC-b1 caused significant reduction of viral infectivity. Time of addition studies demonstrated that HC-b1 exerted its antiviral action at the early stage of virus replication cycle. The presence of HC-b1 could not effectively inhibit the replication of HSV-1 about 45 min after the penetration period started. The antiviral action of HC-b1 appeared to inhibit the attachment of herpes simplex virus on host cell membrane through interfering with the processes of adsorption and penetration.     

不同灵芝菌株菌丝体乙醇提取物体外抑瘤活性的比较和机理

以人急性早幼粒白血病细胞HL-60细胞株作为模型,用MTT法测定生长抑制率,流式细胞Annexin Ⅴ实验和DNA含量法研究抑瘤机制,比较了8个灵芝Ganoderma lucidum菌株发酵菌丝体乙醇提取物的体外抗肿瘤活性和抑瘤机制。筛选结果得到了能产生高抑瘤活性乙醇提取物的灵芝菌株L5。其对HL-60细胞的抑制率为91.4±0.9%(72小时,125μg/mL); 作用48小时后,13.3%的细胞发生早期凋亡,G0/G1期细胞比例比对照组增加15.9%,而S期细胞比例则下降了8.4%,G2/M期细胞数减少7.6%。本研究证明了灵芝菌丝体乙醇提取物能有效抑制HL-60肿瘤细胞的体外生长,抑制率与菌株相关,其抑瘤机制与细胞G0/G1期阻滞和诱导凋亡有关。     

Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata

This study presents the chemical composition and antiviral activity against herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) of sulfated galactan crude extracts and main fractions obtained from two red seaweeds collected in Brazil, Gymnogongrus griffithsiae and Cryptonemia crenulata. Most of the eighteen tested products, including homogeneous kappa/iota/nu carrageenan and DL-galactan hybrid, exhibited antiherpetic activity with inhibitory concentration 50% (IC50) values in the range 0.5-5.6 microg/ml, as determined in a virus plaque reduction assay in Vero cells. The galactans lacked cytotoxic effects and showed a broad spectrum of antiviral activity against HSV-1 and HSV-2. No direct virus inactivation was observed after virion treatment with the galactans. The mode of action of these compounds could be mainly ascribed to an inhibitory effect on virus adsorption. Most importantly, a significant protection against a murine vaginal infection with HSV-2 was afforded by topical treatment with the sulfated galactans.     

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infection affect the phosphorylation of MAPK and Bcl-2 and these proteins may be the common element linking RIP and viral infection. This study investigated the effect of HSV-1 infection on p38 MAPK and Bcl-2 as well as possible interference by TCS. Results showed that HSV-1 infection induced an elevation of phosphorylated p38 and Bcl-2 in Vero cells, which could be partially blocked by TCS. At the same time, both viral replication and host cells viability were lowered. Viral replication, Vero cell viability, p38 MAPK and Bcl-2 were further reduced with the addition of a p38 MAPK inhibitor (SB203580). This suggested that TCS may interfere with MAPK and Bcl-2 signals generated by infection leading to inhibition of viral replication. In summary, our results demonstrated that HSV-1 infection in Vero cells induced an elevation of p38 MAPK and Bcl-2. TCS suppressed this rise and reduced viral replication. The MAPK family may play a role in the antiviral mechanism of TCS.     

Herpes simplex virus gene expression in transformed cells. I. Regulation of the viral thymidine kinase gene in transformed L cells by products of superinfecting virus.

In this paper we show that the expression of the herpes simplex virus type 1 (HSV-1) gene for thymidine kinase (tk) in HSV-transformed cells is subject to regulation by two viral products synthesized during productive infection of these cells with a tk- mutant of HSV-1. The cell line used in this study is a derivative of tk-deficient mouse L cells that, after exposure to UV-inactivated HSV-1, had acquired the HSV-1 gene for tk (which we term a resident viral gene) and consequently expressed the tk+ phenotype (LVtk+ cells). Productive infection of these cells with HSV-1(tk-) at appropriate multiplicities caused significant enhancement of the viral tk activity. The results of several experiments allow us to conclude that this enhancement was due to increased synthesis of tk specified by the HSV-1 gene resident in the LVtk+ cells and that a specific protein made early after infection with HSV-1(tk-) mediated the enhancement, probably by increasing the production of mRNA from the viral tk gene resident in the LVtk+ cells. Our data also indicate that another HSV-1(tk-) product acted to turn off tk synthesis. The finding that tk activity continued to increase for a longer time after infection of the LVtk+ cells at 2 PFU/cell than after infection at higher multiplicities suggested the synthesis of a product which inhibited tk synthesis and whose concentration reached critical levels earlier at higher multiplicities of infection. Inhibition of DNA synthesis after infection, a treatment that depresses the synthesis of late viral proteins, prolonged the synthesis of tk in LVtk+ cells infected at either 2 or 5 PFU/cell. Infection of the LVtk+ cells with HSV-2(tk-) resulted in only small increases in tk activity, indicating some type specificity in recognition of viral products that can modify the expression of the HSV-1 tk gene resident in these cells.     

Mechanism of action of the suppression of herpes simplex virus type 2 replication by pterocarnin A

The purpose of this study was to investigate the in vitro antiviral properties of pterocarnin A, extracted from the bark of Pterocarya stenoptera (Juylandaceae). Results showed that pterocarnin A exhibited anti-herpes simplex virus (HSV) activity. It had a low selectivity index (SI) value and only possessed some level of cell cytotoxic effect at high antiviral concentrations. Mechanism studies demonstrated that pterocarnin A inhibited herpes simplex virus type 2 (HSV-2) from attaching and penetrating into cells. It also actively suppressed HSV-2 multiplication in Vero cells even when added 12 h after infection. This observation indicated that pterocarnin A affected the late stage(s) of HSV-2 infection cycle. Pterocarnin A also significantly reduced viral infectivity at high concentrations. From these observations, it was concluded that pterocarnin A suppressed both early and late in the replication cycle of HSV-2. The various modes of action of pterocarnin A in interfering with certain steps of viral infection thus merit further investigation.     

Susceptibility of human female primary genital epithelial cells to herpes simplex virus, type-2 and the effect of TLR3 ligand and sex hormones on infection

Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.     

The in vitro immunomodulatory activity of a synthetic brassinosteroid analogue would account for the improvement of herpetic stromal keratitis in mice

Herpes simplex virus type 1 (HSV-1) induces an ocular chronic immunoinflammatory syndrome named herpetic stromal keratitis that can lead to vision impairment and blindness. We have reported that the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one, designated as 2, is a potent antiviral in vitro and reduces the incidence of murine herpetic stromal keratitis, although it does not exert an antiviral effect in vivo. In the present report, we investigated whether brassinosteroid 2 may play a role in the modulation of the response of epithelial and immune cells to HSV-1 infection. Compound 2 blocked HSV-1-induced activation of NF-kappaB by inhibiting its translocation to the nucleus of infected corneal and conjunctival cells in vitro, as well as significantly reduced the secretion of TNF-alpha in infected NHC cells. Conversely, IL-6 production was enhanced by compound 2 after HSV-1 infection in both cell types. The production of these cytokines was considerably reduced in a LPS-stimulated macrophage cell line after treatment with compound 2. In conclusion, brassinosteroid 2 would be playing a modulating effect as an inductor or inhibitor, depending on the cell type involved. The improvement of disease observed in mice could be a balance between both, the immunostimulating and immunosuppressive effects of brassinosteroid 2 in vivo.     

Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum

Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties.The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50% of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were > or =2.07 +/- 0.08 microM for FRhK-4 cells: > or =2.23 +/- 0.11 microM for Vero cells; > or =2.54 +/- 0.18 microM for MA104 cells; and > or =2.70 +/- 0.20 microM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 +/- 0.68%, 14.48 +/- 5.06% and 21.47 +/- 3.74% for HSV-1 (strain KOS); 5.96 +/- 2.51%, 8.75 +/- 3.08% and 17.75 +/- 5.19% for HSV-1 (strain ATCC/VR-733); 5.13 +/- 2.38 %, 8.18 +/- 1.11% and 8.51 +/- 1.94% for poliovirus type 2; 8.30 +/- 4.24%; 13.33 +/- 4.66% and 24.27 +/- 2.18% for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay.