Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Equivalência e avaliação da necessidade de sorologia de controle entre esquemas de pré-exposição à raiva humana / Equivalencia y evaluación de la necesidad de serología de control entre esquemas de pre-exposición a la rabia humana / Equivalence between pre-exposure schemes for human rabies and evaluation of the need for serological monitoring

OBJETIVO: Avaliar a resposta imune humoral do esquema de pré-exposição da raiva humana realizado pelas vias intramuscular e intradérmica e a necessidade de sorologia de controle. MÉTODOS: Estudo de intervenção controlado e randomizado, realizado em São Paulo, SP, em 2004-2005. Foram recrutados 149 voluntários, dos quais 127 (65 intradérmica e 62 intramuscular) completaram o esquema de vacinação e realizaram avaliação da resposta imune humoral dez, 90 e 180 dias após o término da vacinação. Foram considerados dois desfechos para a comparação entre as duas vias de aplicação: a média geométrica do título de anticorpos neutralizantes e a proporção de indivíduos com títulos satisfatórios (> 0,5 UI/mL) em cada momento de avaliação. Foi analisada a associação da resposta humoral com dados antropométricos e demográficos por meio de teste de médias e qui-quadrado com correção de Yates. Após a conclusão do esquema foram feitas a comparação da proporção de soropositivos pelo teste de Kruskall Wallis e a comparação dos títulos médios por análise de variância. RESULTADOS: Os títulos médios de anticorpos foram maiores nos indivíduos que receberam as vacinas por via intramuscular. A percentagem de voluntários com títulos satisfatórios (> 0,5 UI/mL) diminuiu com o tempo em ambos os grupos, porém, no grupo que recebeu as vacinas por via intradérmica, a proporção de títulos satisfatórios no dia 180 variou de 20 por cento a 25 por cento, enquanto pela via intramuscular variou de 63 por cento a 65 por cento. Não se observou associação da resposta imune humoral com as variáveis demográficas ou antropométricas. CONCLUSÕES: A sorologia após a terceira dose pode ser considerada desnecessária em indivíduos sob controle quanto à exposição, uma vez que 97 por cento e 100 por cento dos voluntários vacinados, respectivamente por via intradérmica e pela via intramuscular, apresentaram níveis de anticorpos satisfatórios (> 0,5 UI/mL).

OBJECTIVE: To evaluate the humoral immune response to the pre-exposure schedule of human rabies vaccination through intradermal and intramuscular routes, as well as the need for serological monitoring. METHODS: A randomized and controlled intervention study was carried out in São Paulo, Southeastern Brazil, from 2004-2005. There were 149 volunteers, of which 127 completed the vaccination schedule (65 intradermal and 62 intramuscular) and underwent humoral immune response evaluation at ten, 90 and 180 days post-vaccination. Two outcomes were considered for comparing the two routes of administration: the geometric average of neutralizing antibody titers and the proportion of individuals with satisfactory titers (> 0.5 IU/mL) at each evaluation point. The association of the humoral immune response with anthropometric and demographic data was analyzed through a normal distribution test and a chi-square test with a Yates correction. After completion of the vaccination schedule, the proportion of seropositive results was compared by the Kruskall Wallis test, and the average titers were compared by variance analysis. RESULTS: the average antibody titers were higher in patients who were vaccinated intramuscularly. The percentage of volunteers with satisfactory titers (> 0.5 percent IU/mL) decreased over time in both groups. However, in the group vaccinated intradermally the rate of satisfactory titers on day 180 ranged from 20 percent to 25 percent, while the intramuscular route varied from 63 percent to 65 percent. An association between the humoral immune response and the demographic and anthropometric variables was not observed. CONCLUSIONS: Serology after the third dose can be considered unnecessary in unexposed patients, since 97 percent and 100 percent of volunteers respectively vaccinated by the intradermal and intramuscular route presented satisfactory antibody levels (> 0.5 percent IU/mL).

OBJETIVO: Evaluar la respuesta inmune humoral del esquema de pre-exposición de la rabia humana realizado por las vías intramuscular e intradérmica y la necesidad de serología de control. MÉTODOS: Estudio de intervención controlado y aleatorio, realizado en Sao Paulo, Sureste de Brasil, en 2004-2005. Fueron reclutados 149 voluntarios, de los cuales 127 (65 intradérmica y 62 intramuscular) completaron el esquema de vacunación y realizaron evaluación de la respuesta inmune humoral 10, 90 y 180 días posterior al término de la vacunación. Fueron considerados dos resultados para la comparación entre las dos vías de aplicación: el promedio geométrico del título de anticuerpos neutralizantes y la proporción de individuos con títulos satisfactorios (> 0,5 UI/mL) en cada momento de la evaluación. Fue analizada la asociación de la respuesta humoral con datos antropométricos y demográficos por medio de prueba de medias y chi-cuadrado con corrección de Yates. Posterior a la conclusión del esquema fueron realizadas la comparación de la proporción de seropositivos por la prueba de Kruskall Wallis y la comparación de los títulos promedios por análisis de varianza. RESULTADOS: Los títulos promedios de anticuerpos fueron mayores en los individuos que recibieron las vacunas por vía intramuscular. El porcentaje de voluntarios con títulos satisfactorios (> 0,5 UI/mL) disminuyó con el tiempo en ambos grupos, sin embargo, en el grupo que recibió vacuna por vía intradérmica la proporción de títulos satisfactorios en el día 180 varió de 20 por ciento a 25 por ciento, mientras que por la vía intramuscular varió de 63 por ciento a 65 por ciento. No se observó asociación de la respuesta inmune humoral con las variables demográficas o antropométricas. CONCLUSIONES: La serología posterior a la tercera dosis puede ser considerada innecesaria en individuos bajo control con respecto a la exposición, una vez que 97 por ciento y 100 por ciento de los voluntarios vacunados...