Study of the possible effect of alkaloids obtained from the skin secretion of Rhinella jimi and R. icterica in the penetration of rabies virus in mammalian cells mediated by nicotinic acetylcholine receptors

Abstract

Rabies is an acute infectious disease caused by a virus that affects the central nervous system. The virus replicates at the inoculation site and it is believed that use the nicotinic acetylcholine receptors (nAChR) in the neuromuscular junctions to reach the nerve endings. Many alkaloids and other molecules have been isolated from a large number of amphibians and used in various forms of therapy. By the possible fact that rabies virus infects the cell receptor by an alkaloid receptor, this study aims to test alkaloids extracted from the skin of amphibians (Rhinella sp.) as possible interfering agents (antagonists/inhibitors/competitors) in the process of invasion/penetration of the rabies virus in cell, mediated by acetylcholine receptor. The project will be conducted in partnership with Institute Butantan and Institute Pasteur. The secretion of amphibian will be collected through mechanical stimulation of the parotid glands, filtered through a membrane of molecular cut (3 kDa) is processed and then chromatographically. The estrutural determination of molecules will be held initially by mass spectrometry and nuclear magnetic resonance. In the cytotoxic test of the isolated compounds will be used BHK-21 cell line (Baby Hamster Kidney) and murine neuroblastoma (N2A). The cells are deposited in microplate with 96 holes and incubated for 24 and 48h respectively, by another change of medium containing different dilutions of purified molecules. For the virologic test will be used fixed strain PV (Pasteur Virus) and CVS (Challenge Virus Standard) and the techniques used for the test will be Simplified Fluorescence Inhibition (SFIMT) and Quick Test Fluorescent Focus Inhibition Test (RFFIT). (AU)