Rabies Virus-Neutralizing Antibodies in Free-Ranging Invasive Wild Boars (Sus scrofa) from Brazil.

Rabies, one of the most lethal global zoonoses, affects all mammals. It remains circulating worldwide in sylvatic cycles through terrestrial and airborne reservoirs, and in Brazil, bats are currently the main reservoirs and source of transmission. Wild boars, an important invasive alien species in Brazil, are a proven food source for hematophagous bats and may participate in the Brazilian sylvatic cycle of rabies. We evaluated the presence of this pathogen in hunted wild boars from the São Paulo state using histopathology, the direct fluorescent antibody test (DFA), viral isolation in cell culture (VICC), the rapid fluorescent focus inhibition test (RFFIT), and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results of histopathological, DFA, VICC, and RT-qPCR analysis were negative for all samples; seven serum samples tested positive in the RFFIT, and titers ranged from 0.13 IU/mL to 0.5 IU/mL. The presence of rabies virus-neutralizing antibodies in the studied wild boars suggests the circulation of the virus in these animals. Educative actions directed at hunters should include information on the prevention of this important zoonosis.

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.

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.

Análise do possível efeito de componentes da secreção cutânea de Leptodactylus labyrinthicus como interferentes na penetração do vírus da raiva em células de mamíferos / Analysis of the possible effect of components from Leptodactylus labyrinthicus skin secretion as interferents on the rabies virus penetration in mammalian cells

Rabies is a zoonosis distributed worldwide responsible for approximately 55 thousands deaths per year. It is characterized as a lethal progressive acute encephalitis, caused by a virus (Rabies virus; RABV), which replicates in muscular tissues, with subsequent migration to nerve endings possibly mediated by acetylcholine receptors. Similar to other therapeutic classes, molecules with antiviral effects may be present in different organisms, whose toxins, poisons and secretions have been studied as sources of new bioactive substances. In the amphibian’s case, their skin and skin secretions contain a great diversity and variety of bioactive molecules, such as alkaloids, steroids, peptides and proteins, among others. The skin secretion of the anuran Leptodactylus labyrinthicus contains different molecules with biological activity as leptodactyline, pentadactylin and fallaxin. In face of the necessity of new therapeutic approaches for the rabies treatment, this study evaluated whether molecules obtained from the skin secretion of L. labyrinthicus could interfere on the RABV infection in Baby Hamster Kidney (BHK-21) cells line. The skin secretion of L. labyrinthicus was mechanically obtained by compressing the animal submerged in water. This secretion solution was lyophilized, ressuspended in appropriated buffer and filtered in 10 kilodalton (KDa) cut-off membranes. The content retained in the filter was submitted to electrophoresis in polyacrylamide gel. The filtered content was fractionated by Reversed Phase High Performance Liquid Chromatography (RP-HPLC), using a C18 monolithic column and fractions were assayed for cytotoxic and antiviral activity - in BHK-21 cells. After verifying that no fraction was cytotoxic, the evaluation of the possible effect of these fractions on RABV penetration, Pasteur virus (PV) fixed strain, was assessed through tests based on Rapid Fluorescent Focus Inhibition Test (RFFIT). Electrophoretic analysis showed the presence of proteins in the skin secretion of these anurans. From the sixteen fractions obtained, one fraction was able to reduce PV strain infection in BHK-21 cells. Mass spectrometric analyses showed that this fraction contains an antimicrobial peptide named fallaxin, besides low molecular mass molecules. Analogues of this peptide were chemically synthesized by solid phase and assayed in the same models; however, no activity was observed. The active fraction and the synthetic analogues were tested in association with bufotenin, an alkaloid with antiviral effect obtained from the skin secretion of Rhinella jimi. In the virological tests performed with the association of these molecules complete RABV infection inhibition could be observed, pointing out to a synergistic effect between the antibiotic peptide and the alkaloid.

A raiva é uma zoonose mundialmente distribuída responsável pela morte de aproximadamente 55 mil pessoas por ano. É caracterizada como uma encefalite progressiva aguda e letal, causada por um vírus (Rabies virus; RABV) que se replica nos tecidos musculares, com subsequente migração para terminações nervosas, possivelmente, mediada por receptores de acetilcolina. Assim como em outras classes terapêuticas, moléculas com efeitos antivirais podem estar presentes em diferentes organismos, cujas toxinas, venenos e secreções vêm sendo estudadas como fontes de novas substâncias bioativas. No caso dos anfíbios, a pele e a secreção cutânea destes contêm grande diversidade e variedade de moléculas bioativas, como alcaloides, esteroides, peptídeos e proteínas, entre outros. A secreção cutânea do anuro Leptodactylus labyrinthicus possui diferentes moléculas com atividade biológica, como a leptodactilina, a pentadactilina e a falaxina. Devido à necessidade de novas abordagens terapêuticas para o tratamento da raiva, este estudo avaliou se moléculas obtidas a partir da secreção cutânea de L. labyrinthicus poderiam interferir na infecção do RABV em células da linhagem Baby Hamster Kidney (BHK-21). A secreção cutânea de L. labyrinthicus foi mecanicamente obtida por compressão do animal submerso em água. Esta solução de secreção foi liofilizada, ressuspendida em tampão apropriado e filtrada em membranas de corte molecular de 10 quilodaltons (KDa). O conteúdo retido no filtro foi submetido à eletroforese em gel de poliacrilamida. O conteúdo filtrado foi fracionado por cromatografia líquida de alto desempenho de fase reversa (RP-HPLC), utilizando uma coluna monolítica C18, e as frações foram ensaiadas para atividade citotóxica e antiviral – em células BKH-21. Após a constatação de que nenhuma fração foi citotóxica, a avaliação do possível efeito destas frações na penetração da cepa de vírus fixo Pasteur virus (PV) do RABV foi feita através de testes baseados no Teste Rápido de Inibição de Focos Fluorescentes (RFFIT). A análise eletroforética mostrou a presença de proteínas na secreção cutânea destes anuros. Das dezesseis frações obtidas, uma fração foi capaz de diminuir a infecção da cepa PV nas células BHK21. Análises por espectrometria de massas mostraram que esta fração contém um peptídeo antimicrobiano chamado falaxina, além de moléculas de baixa massa molecular. Análogos deste peptídeo foram quimicamente sintetizados, em fase sólida, e ensaiados nos mesmos modelos, porém não tiveram atividade. A fração ativa e os análogos sintéticos foram testados em associação com a bufotenina, um alcaloide com efeito antiviral obtido a partir da secreção cutânea de Rhinella jimi. Nos testes virológicos realizados com as combinações destas moléculas uma completa inibição da infecção pelo RABV pôde ser observada, sugerindo um efeito sinérgico entre o peptídeo antibiótico e o alcaloide.