Effect of the potent antiviral 1-cinnamoyl-3,11-dihydroxymeliacarpin on cytokine production by murine macrophages stimulated with HSV-2.

The limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV-2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF-α, IFN-γ, NO, IL-6 and IL-10 were increased by CDM used alone or in combination with HSV-2. Besides, CDM not only synergized TNF-α production combined with IFN-γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV-2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina.

Therapeutic effect of meliacine, an antiviral derived from Melia azedarach L., in mice genital herpetic infection.

Since natural products are considered powerful sources of novel drug discovery, a partially purified extract (meliacine) from the leaves of Melia azedarach L., a plant used in traditional medicine in India for the treatment of several diseases, has been studied. Meliacine exhibits a potent antiviral effect against several viruses without displaying cytotoxicity. The purpose of the present study was to evaluate the therapeutic effect of intravaginal administration of meliacine in a mouse model of genital herpetic infection. BALB/c female mice were infected with MS or G strains of Herpes Simplex Virus type 2 and then treated with meliacine topically. An overall protective effect was observed. Animal survival increased, the severity of the disease was reduced, life span was extended and virus shedding in vagina fluids was diminished. In addition, meliacine reduced the amount of virus that migrated to the brain and vaginal fluids presented higher levels of IFN-gamma and TNF-alpha than untreated infected mice. These results indicate that meliacine could be an alternative therapeutic compound against HSV-2 genital infection.

Antiviral effect of a synthetic brassinosteroid on the replication of vesicular stomatitis virus in Vero cells.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against replication of vesicular stomatitis virus (VSV) in Vero cells was investigated. Time-related experiments showed that 6b mainly affects a late event of the virus growth cycle. Virus adsorption, internalisation and early RNA synthesis are not the target of the inhibitory action. Results obtained indicate that the antiviral compound adversely affects virus protein synthesis and viral mature particle formation.

The synergistic effect of IFN-alpha and IFN-gamma against HSV-2 replication in Vero cells is not interfered by the plant antiviral 1-cinnamoyl-3, 11-dihydroxymeliacarpin.

BACKGROUND: Recent studies have shown that gamma interferon (IFN-gamma) synergizes with IFN-alpha/beta to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. Since IFN response represents an early host defense event against viral infection and the fact that treatment with meliacine, a plant antiviral, ameliorate the severity of the herpetic infection in female mice infected intravaginally with HSV-2, we wanted to investigate whether the administration of meliacine to HSV-2 infected mice could altered the homoestasis of IFNs host response. For this purpose we studied the effect of the compound 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), which is the responsible for meliacine antiviral action, on the HSV-2 inhibition exerted by IFN alpha, IFN-gamma or their combination. RESULTS: We have found that like HSV-1, IFN-gamma synergizes with IFN-alpha to inhibit HSV-2 replication in Vero cells. While treatment with IFN-alpha or IFN-gamma alone has weak antiviral action, HSV-2 plaque formation, viral replication and the onset of viral CPE in Vero cells are synergistically inhibited by interferon combination. In addition, CDM treatment contributes to protect cells from virus cytopathic effect and causes a strong inhibition of HSV-2 titer. Moreover, the presence of CDM for 2 h before IFN induction, during the 16 h induction period, only for 24 h after infection or during the complete IFN treatment period, reduces virus yields in an additive way without affecting IFN antiviral action. CONCLUSION: The results reported here indicated that the presence of CDM did not alter the antiviral activity of IFN-alpha, IFN-gamma or the synergism exerted by their combination. As a result we can envision that the administration of CDM in vivo could not affect the biological activity of IFNs, which are so important mediators of the innate resistance to HSV-2 infection.

Antiviral mode of action of a synthetic brassinosteroid against Junin virus replication.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against Junin virus replication in Vero cells was investigated. Time-related experiments showed that 6b mainly affects an early event of virus growth cycle. Neither adsorption nor internalization of viral particles was the target of the inhibitory action. The analysis of the effect of 6b on viral RNA synthesis demonstrated that the presence of the compound adversely affects virus RNA replication by preventing the synthesis of full length antigenomic RNA. Although 6b was most effective the earlier it was added to the cells after infection with JV, a high level of inhibition of JV yield and fusion activity of newly synthesized viral glycoproteins was still detected when the compound was present during the last hours of infection. Therefore, we cannot rule out an inhibitory action of 6b on later events of JV replicative cycle.

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.

Block of vesicular stomatitis virus endocytic and exocytic pathways by 1-cinnamoyl-3,11-dihydroxymeliacarpin, a tetranortriterpenoid of natural origin.

Previously, it has been shown that 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a natural compound isolated from leaf extracts of Melia azedarach L., inhibits the vesicular stomatitis virus (VSV) multiplication cycle when added before or after infection. Here, we have established that the lack of VSV protein synthesis in CDM pre-treated Vero cells is ascribed to the inhibition of an initial step during virus multiplication, although indirect immunofluorescence (IFI) studies confirmed that the binding and uptake of [(35)S]methionine-labelled VSV was not affected by CDM pre-treatment. Instead, our findings revealed that this compound impedes the uncoating of VSV nucleocapsids in pre-treated Vero cells, since the antiviral action of CDM was partially reversed by inducing VSV direct fusion at the plasma membrane, and VSV M protein fluorescence was confined to the endosomes, even 2 h post-internalization. Furthermore, CDM induced cytoplasmic alkalinization, as shown by acridine orange staining, consistent with the inhibition of virus uncoating. Although VSV proteins are synthesized when CDM is added after infection, IFI studies revealed that G protein was absent from the surface of infected cells and co-localized with a Golgi marker. Therefore, CDM inhibits the transport of G protein to the plasma membrane. Taken together, these findings indicate that CDM exerts its antiviral action on the endocytic and exocytic pathways of VSV by pre- or post-treatment, respectively.

Antiherpes virus activities of new 6-19 carbon-bridged steroids and some synthetic precursors.

Three synthetic 6,19-carbon bridged steroids: 3beta,20beta-diacetyloxy-5alpha-chloro-19a(R)-hydroxy-6,19-methanopregnane, 3beta,20beta-diacetyloxy-5alpha-chloro-6,19-methanopregnane, 6,19-methanopregn-4-ene-3,20-dione and four synthetic precursors: 3beta,20beta-diacetyloxy-19-hydroxypregn-5-ene, 3beta,20beta-diacetyloxy-pregn-5-en-19-al, 3beta,20beta-diacetyloxy-19(E)-(methoxymethylidene)-pregn-5-ene and 20beta-acetyloxy-3beta-hydroxy-19(E)-(methoxymethylidene)-pregn-5-ene were tested against herpes virus replication in cell cultures. Several compounds were cytotoxic for stationary cells. Antiviral studies performed with all compounds against HSV-1 indicated a dose-dependent virus susceptibility with selectivity indexes (SI) values in the range 1.7-183.2. Selected compounds were also tested against HSV-2 and the SI values obtained were in the range of 31-273. Attempts to reveal the step of virus multiplication affected by pregnanes were performed with one compound. HSV-1 virus incubation with the compound did not alter the ability of virus particles to infect cells; moreover, neither virus adsorption nor penetration appeared to be affected. The drug must be present during at least the first 7 h of the virus cycle to inhibit more than 90% of virus production. All these results suggest that these novel molecules interfere with an intracellular step of virus multiplication, thus behaving like true antivirals.

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.

An antiviral meliacarpin from leaves of Melia azedarach L.

Bioassay guided purification of the ethyl acetate extract of leaves of Melia azedarach led to the isolation of the limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin, which showed IC50 values of 6 microM and 20 microM for vesicular stomatitis (VSV) and herpes simplex (HSV-1) viruses, respectively. Its structure was established by spectroscopic methods.

Therapeutic action of meliacine, a plant-derived antiviral, on HSV-induced ocular disease in mice.

Ocular herpes simplex virus type-1 (HSV-1) infections remain an important cause of corneal disease which may result in a loss of vision. Meliacine (MA), an antiviral activity present in crude leaf extracts of Melia azedarach L. that inhibits HSV-1 multiplication in vitro, was studied in a murine herpetic stromal keratitis experimental model. Adult Balb/c mice were inoculated with HSV-1 at their corneas after abrasion. MA was administered topically three times a day for 3 consecutive days, beginning at 24 and 96 hr after infection. Infected animals treated or not with MA were monitored for the development of ocular disease by a binocular microscope for 16 days. MA significantly reduced the incidence and the severity of blepharitis, neovascularization and stromal keratitis with respect to untreated infected mice, regardless the schedule of treatment assayed. Histological examination of corneas from MA-treated animals revealed no tissue damage, whereas corneal samples from untreated infected mice showed inflammation, vascularization and necrosis. In uninfected mice treated with MA, we found no evidence of corneal damage and histopathological studies showed no changes in the corneas of these mice. Treatment with MA at 24 hours post-infection (h.p.i.) reduced viral multiplication in the eye by 1-1.5 orders of magnitude. Studies on latency revealed that MA sligthly affected the establishment of a latent infection. Thus, MA proved to exert an antiviral action on the development of herpetic stromal keratitis when supplied by post-treatment. Unexpectedly, treatment with MA after 96h.p.i prevented ocular disease, suggesting an in vivo immunomodulating activity of MA.

Antiviral activity of brassinosteroids derivatives against measles virus in cell cultures.

Twenty-seven brassinosteroid derivatives were tested for antiviral activity against measles virus (MV) via a virus-yield reduction assay. Compounds 6b [(22S,235)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one], 1d [(22R,23R)-2alpha,3alpha,22,23-tetrahydroxy-beta-Homo-7-oxa-stigmastan-6-one], 8a [(22R,23R)-3beta-fluoro-22,23-dihydroxystigmastan-6-one], 9b [(22S,23S)-3beta-fluoro-5alpha,22,23-trihydroxystigmastan-6-one] and 10b [(22S,23S)-5alpha-fluor-3beta,22,23-trihydroxystigmastan-6-one], with selectivity indexes (SI) of 40, 57, 31, 37 and 53, are the derivatives with good antiviral activity against MV. These SI values are higher than those obtained with ribavirin (used as reference drug). A comparative analysis of 50% cytotoxic concentration (CC50) values, using confluent non-growing cells, gives and indication of structure-activity relationship. According to their degree of cytotoxicity the compounds were divided in three groups: low, intermediate and high cytotoxicity. By observing the chemical structures of compounds belonging to the first group we can see that less cytotoxic activities are related to the presence of a 3beta-hydroxy group on C-3 (ring A) and a double bond between C-22 and C-23 (side chain). The replacement of a 5alpha-hydroxy group by a 5alpha-fluoro group enhances cytotoxicity. Halogenated brassinosteroid derivatives in C-3 position are more cytotoxic than those with an acetoxy group in the same position. For compounds 1d, 6b, 10b and ribavirin, cytotoxicity measurements were also done with replicating cells; CC50 values were low, but they still competed favourably with ribavirin against MV.

An antiviral principle present in a purified fraction from Melia azedarach L. leaf aqueous extract restrains herpes simplex virus type 1 propagation.

Meliacine (MA), an antiviral principle isolated from leaves of Melia azedarach L., exhibits potent antiviral activity against herpes simplex virus type 1 (HSV-1) by inhibiting specific infected-cell polypeptides (ICPs) produced late in infection. Some of these are involved in DNA synthesis and in the assembly of nucleocapsids. The present report provides additional evidence to elucidate the mode of action of MA against HSV-1. Time-of-addition experiments confirmed that MA affects a late event in the multiplication cycle of HSV-1. We showed that MA diminished the synthesis of viral DNA and inhibited the spread of infectious viral particles when HSV-1 that expresses beta-galactosidase activity was used. In addition, the lack of a protein with an apparent MW of 55 KD was detected in MA-treated cell extracts. Ultrastructural analysis of infected cells showed that, in the case of MA treatment, a large number of unenveloped nucleocapsids accumulated in the cytoplasm and a minor proportion of mature virus was found in cytoplasmic vesicles.These findings suggest that MA exerts an antiviral action on both the synthesis of viral DNA and the maturation and egress of HSV-1 during the infection of Vero cells.

Aspectos éticos que plantea la investigación biomédica básica / Ethical aspects that create the basic biomedical research

En este artículo se señalan y discuten aspectos éticos de la investigación básica de naturaleza biomédica, considerando todo el proces que va desde la formulación de una hipótesis hasta la publicación y utilización de los resultados obtenidos. Dada la complejidad creciente de las investigaciones, en los últimos años se han producido cambios notables en su ejecución, como el carácter multidisciplinario y en consecuencia multiautoral, el elevado costo, el financiamiento por empresas privadas y la necesidad de abordar el patentamiento de los resultados. Lo anterior ha producido la aparición de nuevas situaciones que son imprescindibles de considerar con un enfoque ético. Se analizan también los conflictos de interés que se le presentan al investigador formado contemporáneo, en sus múltiples roles de formador de recursos humanos, asesor en entidades de financiamiento y decisión de prioridades en la investigación, o como revisor de manuscritos a publicar. Se exponen además definiciones de inconducta científica aceptadas por la comunidad de investigadores, pero aún sujetas a debate para lograr una mayor adecuación a los problemas éticos que se presentan dentro de la siempre cambiante investigación biomédica