Plant and animal steroids a new hope to search for antiviral agents.

Scientific literature provides evidence about the use of steroids as an adjunct treatment to antiviral therapies. Immunomodulatory activity of some steroids would account for the recovery in patients with herpetic and other viral infections. However, in vitro studies have demonstrated a direct antiviral effect of this kind of molecules. In this review we discuss recent reports about the mechanism of antiviral action of steroids from animal and plant origin. Chemical structures of most active compounds are also provided.

Effect of meliacine, a plant derived antiviral, on tumor necrosis factor alpha.

The effect of meliacine (MAS) and two fractions MAB 1 and MAB 2 obtained from it on the in vitro production of TNF-alpha of murine macrophages induced by bacterial lipopolysaccharide (LPS) (from Escherichia coli) was tested. Simultaneous administration of the above fractions (ranging from 14 to 56 microg/ml) and LPS (10 microg/ml) to a macrophage culture significantly increased the amount of TNF-alpha released at 24 h of induction in a dose-dependent manner. Meliacine alone, at a concentration of 56 microg/ml, is a weak inducer of TNF-alpha production.

Involvement of vacuolar proton ATPase in Junin virus multiplication.

The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.

Treatment with meliacine, a plant derived antiviral, prevents the development of herpetic stromal keratitis in mice.

Herpetic stromal keratitis is caused by ocular infection with herpes simplex virus type 1 (HSV-1) and constitutes a leading cause of human blindness. The effect of meliacine, an antiviral compound isolated from leaves of Melia azedarach L. that inhibits HSV-1 replication in vitro, was examined on experimental corneal HSV-1 inoculation in Balb/c mice. Mice were inoculated with HSV-1 strain KOS at their corneas after abrasion. Meliacine was administered topically 3 times a day for 4 days beginning 1 day before inoculation. Infected animals treated or not with meliacine were observed carefully for the development of stromal keratitis and the clinical scoring was done 14 days post-infection. Histological examination of corneas and viral isolation from eyes from HSV-1 infected mice treated or not with meliacine were also carried out. It was found that the treatment of HSV-1-induced ocular disease in Balb/c mice with meliacine reduced significantly the development of clinical disease, as well as the histological damage in corneas. The viral titers detected in eyes of infected and treated mice were 2-orders-of-magnitude lower than those corresponding to HSV-1 infected control animals. Mock-infected and treated mice did not reveal any corneal alteration due to the administration of the compound. Meliacine was found to exert a strong antiviral action on HSV-1-induced ocular disease in mice with no evidence of toxic effects.

Antiviral effect of brassinosteroids against herpes virus and arenaviruses.

A natural brassinosteroid and a series of synthetic derivatives were found to be good inhibitors of herpes simplex virus type 1 (HSV-1) and arenavirus replication in cell culture. The synthetic compounds tested were analogues of the 24(S) ethylbrassinone. Compounds (22 R,23 R,24S)-2alpha, 3alpha,5alpha,22,23-pentahydroxy-stigmastan-6-one and (22R,23R,24S)-3beta-bromo-5alpha,22,23-trihydroxy stigmastan-6-one were cytotoxic at concentrations of 20-40 microM. (22S,23S,24S)-2alpha,3alpha,22,23-tetrahydroxy-5alpha, stigmastan-6-one, (22R,23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-choles tan-6-one, (22S,23S,24S)-3beta-bromo-22,23-dihydroxy-5alpha-cholestan-6 -one and (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one were the most active of the series against HSV-1, with selectivity index (SI) values (CC50/EC50) ranging from 10.6 to 16.5. The majority of the compounds were potent inhibitors of arenaviruses, (22S,23S,24S)-3beta-bromo-5alpha,22,23-trihydroxy-stigmastan -6-one being the most active, with SI values of 307.8 and 692.5 for Tacaribe and Junin viruses, respectively. The antiviral activity of brassinosteroid derivatives was not because of direct inactivation; time-of-addition experiments suggested that a late step in HSV-1 multiplication was affected, whereas arenaviruses remained susceptible to the compounds throughout the replicative cycle.

Metabolitos con actividad antiviral aislados de Curvularia pallescens / Antiviral activity metabolites isolated from Curvularia pallescens

The strain BAFC 2336 of Curvularia pallescens is a hyphomycete isolated from internal fungi present in leaves and stems of Baccharis coridifolia. Three compounds designated B, D1 and D2 which inhibited the replication of vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) in tissue cultures were isolated from fluid cultures of Curvularia pallescens. The purification procedure of the compounds consisted first in an organic solvent extraction followed by chromatography through a silica gel column. Fractions eluted from the column with antiviral activity were pooled and then subjected to thin layer chromatography (TLC) in silica gel plates. Three isolated bands were recognized with Rf of 0.63, 0.36 y 0.33 for B, D1 and D2, respectively. The chromatographic characteristics of the isolated metabolites were determined by TLC and HPLC and their chemical structure by means of spectroscopic methods. Analysis of the data obtained indicated that compound D2 (MW: 280 Da) is identical with Brefeldin A a macrolide with antiviral activity isolated from other fungi but not reported to be present in Curvularia pallescens. Compound D1 is similar in structure to compound D2; however, the low amount obtained after purification unabled us to obtain complete structural characterization. Compound B (MW: 332 Da) has an aromatic ring and a chemical structure related to curvularins, a generic name for certain metabolites from Curvularia. This compound appears to be a novel compound with antiviral potency similar to Brefeldin A, but less toxic.(AU)

Metabolitos con actividad antiviral aislados de Curvularia pallescens / Antiviral activity metabolites isolated from Curvularia pallescens

The strain BAFC 2336 of Curvularia pallescens is a hyphomycete isolated from internal fungi present in leaves and stems of Baccharis coridifolia. Three compounds designated B, D1 and D2 which inhibited the replication of vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) in tissue cultures were isolated from fluid cultures of Curvularia pallescens. The purification procedure of the compounds consisted first in an organic solvent extraction followed by chromatography through a silica gel column. Fractions eluted from the column with antiviral activity were pooled and then subjected to thin layer chromatography (TLC) in silica gel plates. Three isolated bands were recognized with Rf of 0.63, 0.36 y 0.33 for B, D1 and D2, respectively. The chromatographic characteristics of the isolated metabolites were determined by TLC and HPLC and their chemical structure by means of spectroscopic methods. Analysis of the data obtained indicated that compound D2 (MW: 280 Da) is identical with Brefeldin A a macrolide with antiviral activity isolated from other fungi but not reported to be present in Curvularia pallescens. Compound D1 is similar in structure to compound D2; however, the low amount obtained after purification unabled us to obtain complete structural characterization. Compound B (MW: 332 Da) has an aromatic ring and a chemical structure related to curvularins, a generic name for certain metabolites from Curvularia. This compound appears to be a novel compound with antiviral potency similar to Brefeldin A, but less toxic.

Antiviral activity of enterocin CRL35 against herpesviruses.

Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) 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 microg/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.

[Antiviral activity metabolites isolated from Curvularia pallescens]. / Metabolitos con actividad antiviral aislados de Curvularia pallescens.

The strain BAFC 2336 of Curvularia pallescens is a hyphomycete isolated from internal fungi present in leaves and stems of Baccharis coridifolia. Three compounds designated B, D1 and D2 which inhibited the replication of vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) in tissue cultures were isolated from fluid cultures of Curvularia pallescens. The purification procedure of the compounds consisted first in an organic solvent extraction followed by chromatography through a silica gel column. Fractions eluted from the column with antiviral activity were pooled and then subjected to thin layer chromatography (TLC) in silica gel plates. Three isolated bands were recognized with Rf of 0.63, 0.36 y 0.33 for B, D1 and D2, respectively. The chromatographic characteristics of the isolated metabolites were determined by TLC and HPLC and their chemical structure by means of spectroscopic methods. Analysis of the data obtained indicated that compound D2 (MW: 280 Da) is identical with Brefeldin A a macrolide with antiviral activity isolated from other fungi but not reported to be present in Curvularia pallescens. Compound D1 is similar in structure to compound D2; however, the low amount obtained after purification unabled us to obtain complete structural characterization. Compound B (MW: 332 Da) has an aromatic ring and a chemical structure related to curvularins, a generic name for certain metabolites from Curvularia. This compound appears to be a novel compound with antiviral potency similar to Brefeldin A, but less toxic.

In vitro anti-Junin virus activity of a peptide isolated from Melia azedarach L. leaves.

Meliacine, a peptide isolated from leaves of Melia azedarach L. inhibited the multiplication of Junin virus in Vero cells treated with the compound before infection (pre-treatment) or immediately after virus adsorption. Analysis of early events following infection demonstrated that meliacine blocks virus penetration by preventing the uncoating step. The addition of meliacine at different times after infection indicated that meliacine also interferes with the release of infectious particles to the extracellular medium and inhibits the low-pH-induced fusion of infected cells. Intracellular transport of viral glycoproteins to the cell membrane was not affected by meliacine, as revealed by immunofluorescence staining. Taken together, these results suggest that meliacine affects two events of the virus replicative cycle that require membrane fusion: uncoating and budding.

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves.

Meliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 degrees C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification.

In vitro and in vivo activities of Melia azedarach L. aqueous leaf extracts on murine lymphocytes.

The effect of Melia azedarach L. aqueous leaf extracts (Ma) on in vitro lymphocyte proliferation and humoral and cellular immune responses in vivo was studied. Proliferation of spleen and lymph node T cells was impaired when these cells were incubated in the presence of the extract using different mitogens as stimuli. Furthermore, treatment of mice with the extracts not only diminished the production of antibodies but also exerted an inhibition on graft vs host and delayed type hypersensitivity reactions. These results suggest that Ma extracts exert a marked immunomodulatory effect on the mouse immune system.

Antiviral activity of meliacine on the replication of a thymidine kinase-deficient mutant of Herpes simplex virus type 1 alone and in combination with acyclovir.

The anti-HSV-1 activity of meliacine (MA), a peptide isolated from leaves of Melia azedarach L., alone and in combination with acyclovir (ACV), was assayed against thymidine kinase-deficient (TK(-)) virus yields in vitro. MA alone proved to inhibit significantly TK(-) viral replication, whereas ACV was more potent than MA as an inhibitor of TK(+) replication. TK(-) and TK(+) synergistic inhibition by the combination of both agents was observed at concentrations that did not alter cell viability. The interaction between MA and ACV was quantitatively determined by calculating the combination index and plotting the data by the isobologram method. Besides, MA and ACV were able to suppress synergistically the antigen expression on HSV-I infected cells processed by an immunofluorescence assay. These in vitro findings suggest that combinations of MA and ACV at appropriate doses may provide an increased efficacy in inhibiting both TK(-) and TK(+) HSV-1 multiplication.

Herpes simplex virus-inhibitory sulfated xylogalactans from the red seaweed Nothogenia fastigiata.

Two sulfated xylogalactans (F1 and F7), isolated from the red seaweed Nothogenia fastigiata, achieved a dose-dependent inhibition of the replication of herpes simplex virus type 1 (HSV-1) in Vero cells, with 50% effective doses in the range of 15.0-32.6 micrograms/ml, and without affecting cell viability at concentrations up to 200 micrograms/ml. The presence of sulfate groups in the molecule was essential for the antiviral properties of these polysaccharides. F7 afforded significant inhibition in HSV-1 yield if added to the cell cultures simultaneously with virus inoculum, but had no effect when it was added after 1 h of infection. Analysis of the early events of the viral replicative cycle showed that the anti-HSV effect of F7 was due to a specific inhibition of virus attachment to the host cell whereas virus internalization was not impaired.

Determinación de la actividad antiviral de un xilomanano sulfatado de origen natural en distintas condiciones experimentales / Determination of the antiviral activity of a natural sulphated xylomannan under different experimental conditions

Se analizó la influencia de distintas condiciones experimentales sobre la determinación de la actividad antiviral del xilomanano sulfatado F6, aislado del alga roja patagónica Nothogenia fastigiata, sobre la multiplicación de los virus herpes simplex tipos 1 y 2 (HSV-1 y HSV-2). En las condiciones ensayadas, la concentración inhibitoria 50 por ciento (CI50) de F6 resultó independiente de la multiplicidad de infección (en el rango 0,1-0,0001 UFP/célula), del tipo de ensayo antiviral empleado (inhibición del rendimiento viral o reducción en la formación de placas), de la cepa de virus (F, KOS, B2006, Field) y de la línea celular (Vero, HEp-2, BHK-21). La independencia del efecto inhibitorio de F6 respecto de la multiplicidad de infección representa una ventaja para este polisacárido respecto de otros compuestos antivirales que sólo son activos frente a muy bajas dosis de virus (AU)

Determinación de la actividad antiviral de un xilomanano sulfatado de origen natural en distintas condiciones experimentales / Determination of the antiviral activity of a natural sulphated xylomannan under different experimental conditions

Se analizó la influencia de distintas condiciones experimentales sobre la determinación de la actividad antiviral del xilomanano sulfatado F6, aislado del alga roja patagónica Nothogenia fastigiata, sobre la multiplicación de los virus herpes simplex tipos 1 y 2 (HSV-1 y HSV-2). En las condiciones ensayadas, la concentración inhibitoria 50 por ciento (CI50) de F6 resultó independiente de la multiplicidad de infección (en el rango 0,1-0,0001 UFP/célula), del tipo de ensayo antiviral empleado (inhibición del rendimiento viral o reducción en la formación de placas), de la cepa de virus (F, KOS, B2006, Field) y de la línea celular (Vero, HEp-2, BHK-21). La independencia del efecto inhibitorio de F6 respecto de la multiplicidad de infección representa una ventaja para este polisacárido respecto de otros compuestos antivirales que sólo son activos frente a muy bajas dosis de virus

[Determination of the antiviral activity of a naturally occurring sulfated xylomannan under various experimental conditions]. / Determinación de la actividad antiviral de un xilomanano sulfatado de origen natural en distintas condiciones experimentales.

The effects of different experimental conditions on the antiviral activity of the sulphated xylomannan F6, isolated from the red seaweed Nothogenia fastigiata, against the replication of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) were studied. The compound was equally effective against several strains of HSV-1 (F, KOS, B-2006 and Field) and HSV-2 (G) in different cell lines (Vero, HEp-2 and BHK-21). Furthermore, the antiviral activity of F6 was independent of the method employed to determine the 50% inhibitory concentration (IC50) (virus yield or plaque reduction tests). The value of the IC50 against HSV-1 was not significantly affected by the initial virus concentration since a variation in the multiplicity of infection from 0.0001 to 0.1 UFP/cell only increased twofold the IC50 from 0.61 to 1.35 micrograms/ml, respectively. The potent inhibitory effect of F6 against high doses of HSV-1 represents a significant advantage for this sulphated polysaccharide with respect to other compounds only active against low viral inocula.

Susceptibilidad de Picornavirus a un antiviral de origen vegetal (Meliacina) / Susceptibility of picornaviruses to an antiviral of vegetal origin (meliacine)

La planta superior Melia azederach L (MA) produce un polipéptido cíclico que puede aislarse de sus hojas y que tiene un amplio espectro antiviral contra virus con RNA de distintas familias. En este trabajo se estudió su efecto sobre dos Picornavirus: el virus fiebre aftosa (VFA) y el virus polio. Para ello se probaron las cepas A24, A87, C3 Resende, O1 Campos, O1 Caseros y O69 del VFA y los tipos 1, 2 y 3 del virus polio. Se encontró que las cepas muestran una susceptibilidad diferencial a una concentración no citotóxica del antiviral de 0,3 Ag/ml, siendo las cepas A24 y A87 las más susceptibles ya que resultaron inhibidas en un 90 por ciento. Para que ello ocurra el extracto de MA debe agregarse después de la adsorción viral y conservarse en el medio de cultivo hasta la cosecha de virus ya que con todas las cepas ensayadas el pretratamiento de las monocapas no resultó efectivo. Para determinar la influencia de la multiplicidad de infección (m.i.) en la diferente susceptibilidad observada se eligió la cepa O1 Campos del VFA y la tipo 3 de polio que resultaron ser poco sensibles cuando se usó una m.i. de 1, encontrándose una inhibición del 99 por ciento para ambos virus a una m.i. de 0,001. Estos resultados indican que los Picornavirus también son susceptibles a la inhibición por meliacina, que dicha inhibición varía con la cepa ensayada y que la aparente resistencia de estos virus estaría relacionada con la velocidad del ciclo de replicación, el que es superior al establecimiento del estado antiviral, cuando todas las células son infectadas simultáneamente (AU)

Susceptibilidad de Picornavirus a un antiviral de origen vegetal (Meliacina) / Susceptibility of picornaviruses to an antiviral of vegetal origin (meliacine)

La planta superior Melia azederach L (MA) produce un polipéptido cíclico que puede aislarse de sus hojas y que tiene un amplio espectro antiviral contra virus con RNA de distintas familias. En este trabajo se estudió su efecto sobre dos Picornavirus: el virus fiebre aftosa (VFA) y el virus polio. Para ello se probaron las cepas A24, A87, C3 Resende, O1 Campos, O1 Caseros y O69 del VFA y los tipos 1, 2 y 3 del virus polio. Se encontró que las cepas muestran una susceptibilidad diferencial a una concentración no citotóxica del antiviral de 0,3 µg/ml, siendo las cepas A24 y A87 las más susceptibles ya que resultaron inhibidas en un 90 por ciento. Para que ello ocurra el extracto de MA debe agregarse después de la adsorción viral y conservarse en el medio de cultivo hasta la cosecha de virus ya que con todas las cepas ensayadas el pretratamiento de las monocapas no resultó efectivo. Para determinar la influencia de la multiplicidad de infección (m.i.) en la diferente susceptibilidad observada se eligió la cepa O1 Campos del VFA y la tipo 3 de polio que resultaron ser poco sensibles cuando se usó una m.i. de 1, encontrándose una inhibición del 99 por ciento para ambos virus a una m.i. de 0,001. Estos resultados indican que los Picornavirus también son susceptibles a la inhibición por meliacina, que dicha inhibición varía con la cepa ensayada y que la aparente resistencia de estos virus estaría relacionada con la velocidad del ciclo de replicación, el que es superior al establecimiento del estado antiviral, cuando todas las células son infectadas simultáneamente