Crotoxin and phospholipases A₂ from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses

Abstract

Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic fever with high mortality that is transmitted by mosquitoes. 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. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. 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 inhibition of Yellow fever and dengue viruses in VERO E6 cells.

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.