Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses

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 A(2) showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells. (C) 2011 Elsevier Ltd. All rights reserved.Sao Paulo Research Foundation (FAPESP

Phospholipase A₂ Isolated from the Venom of <i>Crotalus durissus terrificus</i> Inactivates <i>Dengue virus</i> and Other Enveloped Viruses by Disrupting the Viral Envelope

<div><p>The <i>Flaviviridae</i> family includes several virus pathogens associated with human diseases worldwide. Within this family, <i>Dengue virus</i> is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against <i>Dengue virus</i> or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A₂ isolated from the venom of <i>Crotalus durissus terrificus</i> was able to inhibit <i>Dengue virus</i> and <i>Yellow fever virus</i> infection in Vero cells. Here, we present evidence that phospholipase A₂ has a direct effect on <i>Dengue virus</i> particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A₂ might gain access to the <i>Dengue virus</i> lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A₂ is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.</p></div

Analysis of the exposure of DENV-2 RNA.

<p>DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p<0.01).</p

Analysis of the exposure of DENV-2 genomic RNA.

<p>DENV-2 was first treated with PLA₂-CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p<0.05).</p

Pre-treatment assay.

<p>Vero cells were treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then infected with DENV-2 for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated cells (*p<0.05, **p<0.01).</p

Virucidal assay.

<p>DENV-2 was treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then used to infect Vero cells for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (*<i>p</i><0.05, **p<0.01).</p

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 mu g/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox`s lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2005/54855-0

Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP