title: Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus.
abstract: 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.
authors.affiliation: Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil ; Pasteur Institute, Laboratory of Rabies Diagnostic, Serology, Avenida Paulista, 393, São Paulo, 01311-000 SP Brazil.
authors.forename: Rene Dos Santos
authors.initials: Rdos S
authors.lastname: Cunha Neto
authors.affiliation: Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil.
authors.forename: Hugo
authors.initials: H
authors.lastname: Vigerelli
authors.affiliation: Butantan Institute, Laboratory of Cell Biology, Av Vital Brasil, 1500, São Paulo, 05503-900 SP Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Jared
authors.affiliation: Butantan Institute, Laboratory of Cell Biology, Av Vital Brasil, 1500, São Paulo, 05503-900 SP Brazil.
authors.forename: Marta Maria
authors.initials: MM
authors.lastname: Antoniazzi
authors.affiliation: Pasteur Institute, Laboratory of Rabies Diagnostic, Serology, Avenida Paulista, 393, São Paulo, 01311-000 SP Brazil.
authors.forename: Luciana Botelho
authors.initials: LB
authors.lastname: Chaves
authors.affiliation: Pasteur Institute, Laboratory of Rabies Diagnostic, Serology, Avenida Paulista, 393, São Paulo, 01311-000 SP Brazil.
authors.forename: Andréa de Cássia Rodrigues
authors.initials: Ade C
authors.lastname: da Silva
authors.affiliation: Butantan Institute, Special Laboratory of Toxinology, Av Vital Brasil, 1500, São Paulo, 05503-900 SP Brazil.
authors.forename: Robson Lopes de
authors.initials: RL
authors.lastname: Melo
authors.affiliation: Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil.
authors.forename: Juliana Mozer
authors.initials: JM
authors.lastname: Sciani
authors.affiliation: Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil.
authors.forename: Daniel C
authors.identifier: 0000-0003-2406-0860
authors.initials: DC
authors.lastname: Pimenta
keywords: Bufotenine; Leptodactylus; Ocellatin-F1; Rabies virus; Toxin