Crotoxin and phospholipases A2 from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses
Highlights
► Antiviral activity of C.d. terrificus venom was evaluated against DENV and YFV. ► The PLA2 showed high activity in pre-treatment, virucidal and adsorption assays. ► The catalytic activity of PLA2 is important for antiviral effect.
Introduction
Dengue virus (DENV) and yellow fever virus (YFV), members of the genus Flavivirus, family Flaviviridae, are two of the most important arboviruses in public health (Gould and Solomon, 2008). Dengue is the most rapidly spreading arbovirus disease in the world. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. An estimated 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries (WHO, 2010). Infection with any of the four DENV serotypes (DENV-1, -2, -3 and -4) can be asymptomatic or can lead to a wide spectrum of disease, in some cases with fatal outcome (Harris et al., 2000). YFV is the causative agent of severe acute hemorrhagic fever with high mortality. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa (Robertson et al., 1996, World Health Organization (WHO), 2010). There is no specific drug therapy for DENV and YFV infections; therefore, the development of antiviral agents to reduce the morbidity and mortality causes by these two viruses is a public health priority (Hombach et al., 2005).
Snake venoms are complex mixtures of toxins and enzymes that show different activities on biological systems, such as cytotoxicity, hemorrhage activity, bradykinin-releasing activity, thrombin-like activity, hemolysis, cardiovascular and hypotensive effects, tissue necrosis and neurotoxic effects (Castro et al., 2004, Debnath et al., 2010, Gutiérrez et al., 2005, Lee, 1977, Montecucco et al., 2008, Oyama et al., 2008, Vital-Brazil, 1982). The venom of Crotalus durissus terrificus snake, a South American rattlesnake, has shown several biological activities, including antiviral activity against measles virus (Bercovici et al., 1987, Petricevich and Mendonça, 2003). This venom is composed by neurotoxins, crotoxin (Slotta and Fraenkel-Conrat, 1938), crotamin (Gonçalves and Vieira, 1950), phospholipase A2 “inter-cro” (PLA2-IC) (Vieira, 2009), gyroxin (Alexander et al., 1988) and convulxin (Prado-Franceschi and vital Brazil, 1981). The fraction which pathophysiological aspects are better characterized is the crotoxin. It represents 40–60% of the dry weight of venom and it is the main toxic component with neurotoxic effects (Faure and Bon, 1988, Vital-Brazil, 1966). This component presents two different subunits no covalently linked: crotapotin, an acid component with a molecular weight of ∼9,000 Da and the phospholipase A2, a basic component (PLA2-CB) with a molecular weight of 16,400 Da (Hendon and Fraenkel-Conrat, 1971, Rubsamen et al., 1971). Faure et al. (1994) isolated and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Crotoxin is, in fact, a mixture of variants deriving from the combination of subunit isoforms. Four crotapotin and four PLA2-CB present in venom collected from numerous snakes were purified and some sequenced (Faure et al., 1991). The crotapotin isoforms consist of three disulfide-linked polypeptide chains (α, β, γ ), which result from different proteolytic cleavages of a unique precursor pro-crotapotin that has been identified from its cDNA. Two cDNAs encoding PLA2-CB isoforms have been cloned and their nucleic acid sequences determined (Bouchier et al., 1991). The PLA2-IC was first observed by Laure (1975), but the enzyme was not isolated or characterized. Vieira (2009) subsequently isolated and characterized the PLA2-IC demonstrating that was a new isoform, showing differences in the C-terminal region when compared with PLA2-CB1 and PLA2-CB2 (basic chain of crotoxin). The alignment of the PLA2-CB1, PLA2-CB2 and PLA2-IC sequences and phylogenetic analysis showed that PLA2-CB2 isoform showed higher homology with the PLA2-IC isoform and, furthermore, this homology is greater than that observed between PLA2-CB1 and PLA2-IC and even between PLA2-CB1 and PLA2-CB2. The PLA2-CB2 was originated from the duplication of the PLA2-CB1 gene (Vieira, 2009). Faure et al. (1994) demonstrated that the complex PLA2-CB1/CA present in crotoxin, is more stable than PLA2-CB2/CA association, confirming that PLA2-IC is an isoform of PLA2-CB2, which does not make the association with crotapotin but is able to exert its biological activity present in the venom of C. d. terrificus.
In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A2 showed a high antiviral effect against DENV and YFV.
Section snippets
Cells and viruses
VERO E6 and C6/36 cells were maintained in Leibovitz medium (L-15) with 10% of fetal bovine serum (FBS) at 37 °C and 28 °C, respectively. DENV-2 (strain NGC) and YFV (strain 17D) were used in this study. The viruses were propagated in C6/36 cells, titrated by plaque formation assay in VERO E6 cells and expressed in plaque forming units per milliliters (PFU/mL).
Venom and isolated toxins
Lyophilized yellow and white crude venom from C. d. terrificus and crude venom from Bothrops jararacussu were obtained from the
Purification of toxins from C. d. terrificus and B. jararacussu
The venom of C. d. terrificus was subjected to size exclusion chromatography on Sephadex G-75 to obtain the fractions containing convulxin (I), giroxin (II), crotoxin (III), PLA2-IC (IV), crotamin (V) and other peptides (VI) (Fig. 1A). After the first chromatographic step, crotoxin fraction was subjected to a cationic exchange chromatography to obtain crotapotin (CA) and PLA2-CB (CB) (Fig. 1B). The purity of the purified proteins was estimated by SDS-PAGE (Fig. 1C).
In Fig. 1C, crotamim,
Discussion
Several studies describe the use of natural or synthetic substances as potential antidengue or antiyellow fever agents (Benarroch et al., 2004, Poh et al., 2009, Talarico et al., 2005, Wang et al., 2009, Zhang et al., 2009). However, currently there is no drug available for treatment of these infections.
Snake venoms have shown to present antibacterial (Samy et al., 2010, Wang et al., 2009), antiparasite (Deolindo et al., 2010), antifungal (Magaldi et al., 2002), and antiviral activities (Fenand
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was funded by the Sao Paulo Research Foundation (FAPESP).
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These authors contributed equally.