Análise do possível efeito de componentes da secreção cutânea de Leptodactylus labyrinthicus como interferentes na penetração do vírus da raiva em células de mamíferos

Abstract

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.