Viperid Snakes Research Articles

Interrogating the Venom of the Viperid Snake Sistrurus catenatus edwardsii by a Combined Approach of Electrospray and MALDI Mass Spectrometry.

The complete sequence characterization of snake venom proteins by mass spectrometry is rather challenging due to the presence of multiple isoforms from different protein families. In the present study, we investigated the tryptic digest of the venom of the viperid snake Sistrurus catenatus edwardsii by a combined approach of liquid chromatography coupled to either electrospray (online) or MALDI (offline) mass spectrometry. These different ionization techniques proved to be complementary allowing the identification a great variety of isoforms of diverse snake venom protein families, as evidenced by the detection of the corresponding unique peptides. For example, ten out of eleven predicted isoforms of serine proteinases of the venom of S. c. edwardsii were distinguished using this approach. Moreover, snake venom protein families not encountered in a previous transcriptome study of the venom gland of this snake were identified. In essence, our results support the notion that complementary ionization techniques of mass spectrometry allow for the detection of even subtle sequence differences of snake venom proteins, which is fundamental for future structure-function relationship and possible drug design studies.

Cytotoxic Effect of Iranian Vipera lebetina Snake Venom on HUVEC Cells.

Envenomation by heamotoxic snakes constituted a critical health occurrence in the world. Bleeding is the most sever consequence following snake bite with viperid and crothalid snakes. It is believed that the degradation of vascular membrane caused hemorrhage; in contrast, some suggested that direct cytotoxicity has role in endothelial cell disturbances. This study was carried out to evaluate the direct toxicity effect of V. lebetina crude venom on Human Umbilical Vein Endothelial Cells (HUVECs). The effect of V. lebetina snake venom on HUVECs growth inhibition was determined by MTT assay and neutral red uptake assay. The integrity of cell membrane through LDH release was measured with the Cytotoxicity Detection Kit. Morphological changes of endothelial cells were also evaluated using a phase contrast microscope. In MTT assay, crude venom showed a cytotoxic effect on endothelial cells which was confirmed by the effect observed with neutral red assay. Also, crude venom caused changes in the integrity of cell membrane by LDH release. The morphological alterations enhanced in high concentration results in total cells number reduced. V. lebetina venom showed potential direct cytotoxic effects on human endothelial cells in a manner of concentration- dependent inhibition.

Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins.

Current treatment for venom-induced consumption coagulopathy resulting from snakebite.

Venomous snakebite is considered the single most important cause of human injury from venomous animals worldwide. Coagulopathy is one of the commonest important systemic clinical syndromes and can be complicated by serious and life-threatening haemorrhage. Venom-induced consumption coagulopathy (VICC) is the commonest coagulopathy resulting from snakebite and occurs in envenoming by Viperid snakes, certain elapids, including Australian elapids, and a few Colubrid (rear fang) snakes. Procoagulant toxins activate the clotting pathway, causing a broad range of factor deficiencies depending on the particular procoagulant toxin in the snake venom. Diagnosis and monitoring of coagulopathy is problematic, particularly in resource-poor countries where further research is required to develop more reliable, cheap clotting tests. MEDLINE and EMBASE up to September 2013 were searched to identify clinical studies of snake envenoming with VICC. The UniPort database was searched for coagulant snake toxins. Despite preclinical studies demonstrating antivenom binding toxins (efficacy), there was less evidence to support clinical effectiveness of antivenom for VICC. There were no placebo-controlled trials of antivenom for VICC. There were 25 randomised comparative trials of antivenom for VICC, which compared two different antivenoms (ten studies), three different antivenoms (four), two or three different doses or repeat doses of antivenom (five), heparin treatment and antivenom (five), and intravenous immunoglobulin treatment and antivenom (one). There were 13 studies that compared two groups in which there was no randomisation, including studies with historical controls. There have been numerous observational studies of antivenom in VICC but with no comparison group. Most of the controlled trials were small, did not use the same method for assessing coagulopathy, varied the dose of antivenom, and did not provide complete details of the study design (primary outcomes, randomisation, and allocation concealment). Non-randomised trials including comparison groups without antivenom showed that antivenom was effective for some snakes (e.g., Echis), but not others (e.g., Australasian elapids). Antivenom is the major treatment for VICC, but there is currently little high-quality evidence to support effectiveness. Antivenom is not risk free, and adverse reactions can be quite common and potentially severe. Studies of heparin did not demonstrate it improved outcomes in VICC. Fresh frozen plasma appeared to speed the recovery of coagulopathy and should be considered in bleeding patients.

Isolation and characterization of four medium-size disintegrins from the venoms of Central American viperid snakes of the genera Atropoides, Bothrops, Cerrophidion and Crotalus.

Four disintegrins were isolated from the venoms of the Central American viperid snakes Atropoides mexicanus (atropoimin), Bothrops asper (bothrasperin), Cerrophidion sasai (sasaimin), and Crotalus simus (simusmin). Purifications were performed by reverse-phase HPLC. The four disintegrins have biochemical characteristics, i.e. molecular mass and location of Cys, which allow their classification within the group of medium-size disintegrins. All of them present the canonical RGD sequence, which determines their interaction with integrins in cell membranes. The disintegrins inhibited ADP and collagen-induced human platelet aggregation, with similar IC50s in the nM range. In addition, disintegrins inhibited the adhesion of an endothelial cell line and a melanoma cell line to the extracellular matrix proteins type I collagen, laminin, fibronectin, and vitronectin, albeit showing variable ability to exert this activity. This study expands the inventory of this family of viperid venom proteins, and reports, for the first time, disintegrins from the venoms of species of the genera Atropoides and Cerrophidion.

Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and macromolecular substrates and indicates that their hydrolytic activity is influenced by the amino acid sequences adjacent to the scissile bond.

Synergism between Basic Asp49 and Lys49 Phospholipase A2 Myotoxins of Viperid Snake Venom In Vitro and In Vivo

Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.

Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes.

We report the discovery of endogenous viral elements (EVEs) from Hepadnaviridae, Bornaviridae and Circoviridae in the speckled rattlesnake, Crotalus mitchellii, the first viperid snake for which a draft whole genome sequence assembly is available. Analysis of the draft assembly reveals genome fragments from the three virus families were inserted into the genome of this snake over the past 50 Myr. Cross-species PCR screening of orthologous loci and computational scanning of the python and king cobra genomes reveals that circoviruses integrated most recently (within the last approx. 10 Myr), whereas bornaviruses and hepadnaviruses integrated at least approximately 13 and approximately 50 Ma, respectively. This is, to our knowledge, the first report of circo-, borna- and hepadnaviruses in snakes and the first characterization of non-retroviral EVEs in non-avian reptiles. Our study provides a window into the historical dynamics of viruses in these host lineages and shows that their evolution involved multiple host-switches between mammals and reptiles.

A new C-type lectin (RVsnaclec) purified from venom of Daboia russelii russelii shows anticoagulant activity via inhibition of FXa and concentration-dependent differential response to platelets in a Ca2 +-independent manner

This is the first report on the characterization of a snaclec (RVsnaclec) purified from Daboia russelii russelii venom. The RVsnaclec is a heterodimer of two subunits, α (15.1kDa) and β (9kDa). These subunits are covalently linked to form multimeric (αβ)2 and (αβ)4 structures. Peptide mass fingerprinting analysis of RVsnaclec via LC-MS/MS demonstrated its similarity to snaclecs purified from other viperid snake venoms. Two tryptic peptide sequences of RVsnaclec revealed the putative conserved domains of C-type lectin (CTL). RVsnaclec dose-dependently increased the Ca-clotting time and prothrombin time of platelet-poor plasma (PPP); however, it did not affect the partial thromboplastin time (APTT) or thrombin time of PPP. The in vitro and in vivo anticoagulant activity of RVsnaclec is correlated to its binding and subsequent uncompetitive inhibition of FXa (Ki=0.52 μmole) in a Ca2+-independent manner; however, supplementation with 0.25mM Ca2+ enhanced the Xa binding potency of RVsnaclec. Monovalent or polyvalent antivenom failed to neutralize its anticoagulant potency, and RVsnaclec did not inhibit trypsin, chymotrypsin, thrombin or plasmin. RVsnaclec was devoid of hemolytic activity or cytotoxicity against several human cancer cell lines, demonstrated concentration-dependent aggregation and deaggregation of human platelets, and inhibited the ADP-induced aggregation of platelet. RVsnaclec (5.0mg/kg body weight) was non-lethal to mice and showed no adverse pharmacological effects, suggesting that it has potential as a lead compound for future therapeutic applications in cardiovascular disorders.

Seasonal, daily activity, and habitat use by three sympatric pit vipers (Serpentes, Viperidae) from southern Brazil.

Viperid snakes are widely distributed in the South America and the greater distribution range of the family is found at the Crotalinae subfamily. Despite the abundance of this snakes along their geographic distribution, some ecological aspects remain unknown, principally at subtropical areas. In the present study, we evaluated the activity (daily and seasonal) and the use of the habitat by Bothrops diporus, B. jararaca and B. jararacussu, in an Atlantic Forest area at southern Brazil. We observed higher incidence of viperid snakes during the months with higher temperatures, while no snakes were found during the months with lower temperatures. The data suggest the minimum temperature as environmental variable with the greatest influence on the seasonal activity of this species. Considering the daily activity, we observed a tendency of snakes to avoid the warmest hours. Bothrops jararacussu tend to avoid open areas, being registered only inside and at the edges of the forest. We compared our results with previous studies realized at tropical areas and we suggest the observed seasonal activity as an evolutive response, despite the influence of the different environmental variables, according to the occurence region.

Morphology, Reproduction and Diet in Australian and Papuan Death Adders (Acanthophis, Elapidae)

Death adders (genus Acanthophis) differ from most other elapid snakes, and resemble many viperid snakes, in their thickset morphology and ambush foraging mode. Although these snakes are widely distributed through Australia and Papua New Guinea, their basic biology remains poorly known. We report morphological and ecological data based upon dissection of >750 museum specimens drawn from most of the range of the genus. Female death adders grow larger than conspecific males, to about the same extent in all taxa (20% in mean adult snout-vent length, = SVL). Most museum specimens were adult rather than juvenile animals, and adult males outnumbered females in all taxa except A. pyrrhus. Females have shorter tails (relative to SVL) than males, and longer narrower heads (relative to head length) in some but not all species. The southern A. antarcticus is wider-bodied (relative to SVL) than the other Australian species. Fecundity of these viviparous snakes was similar among taxa (mean litter sizes 8 to 14). Death adders encompass a broad range of ecological attributes, taking a wide variety of vertebrate prey, mostly lizards (55%), frogs and mammals (each 21%; based on 217 records). Dietary composition differed among species (e.g. frogs were more common in tropical than temperate-zone species), and shifted with snake body size (endotherms were taken by larger snakes) and sex (male death adders took more lizards than did females). Overall, death adders take a broader array of prey types, including active fast-moving taxa such as endotherms and large diurnal skinks, than do most other Australian elapids of similar body sizes. Ambush foraging is the key to capturing such elusive prey.

Elapid Snake Venom Analyses Show the Specificity of the Peptide Composition at the Level of Genera Naja and Notechis

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms—that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.

Understanding structural and functional aspects of PII snake venom metalloproteinases: Characterization of BlatH1, a hemorrhagic dimeric enzyme from the venom of Bothriechis lateralis

A new homodimeric PII metalloproteinase, named BlatH1, was purified from the venom of the Central American arboreal viperid snake Bothriechis lateralis by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. BlatH1 is a glycoprotein of 84 kDa. The mature protein contains a metalloproteinase domain, with the characteristic zinc-binding motif (HEXXHXXGXXH) followed by the sequence CIM at the Met-turn. In the disintegrin domain, the tripeptide sequence TDN substitutes the characteristic RGD motif found in many disintegrins. BlatH1 hydrolyzed azocasein, gelatin and fibrinogen, and exerts a potent local and systemic hemorrhagic activity in mice. The hemorrhagic activity of BlatH1 is not inhibited by the plasma proteinase inhibitor α2-macroglobulin, although the SVMP is able to cleave this plasma inhibitor, generating a 90 kDa product. BlatH1 inhibits ADP- and collagen-induced human platelet aggregation (IC50 = 0.3 μM and 0.7 μM for ADP and collagen, respectively). This activity is abrogated when the enzyme is preincubated with the metalloproteinase inhibitor Batimastat, implying that it depends on proteolysis. In agreement, a synthetic peptide containing the sequence TDN of the disintegrin domain is unable to inhibit platelet aggregation. BlatH1 is a valuable tool to understand the structural determinants of toxicity in PII SVMPs.

Risk Factors Associated with Bites due to Two Viperid Snakes: A Case Control Study

Aims: This study was to assess victim’s environmental and behavioural risk factors that promote bites from two viperid snakes. Study Design: A case control study.

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

In this research a preliminary identification and biochemical and biological characterization of a PLA2 (Btae TX-I) from the venom of a viperid snake, Bothriopsis taeniata (Speckled forest pit viper) were obtained. Btae TX-I was purified by two chromatographic steps, molecular exclusion chromatography followed by analytical chromatography reverse phase HPLC. Molecular mass behaved as a homogeneous single chain protein on SDS–PAGE, confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 13889.98 Da. Tryptic peptides were determined in tandem mass spectrometry and showed similarity with other myotoxic PLA2s. Btae TX-I belongs to the Asp49 PLA2 class, is enzymatically active in presence of a synthetic substrate and shows a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45°C. PLA2 activity in presence of Mn2+, Mg2+, Cd2+ and Zn2+ was reduced either in presence or absence of Ca2+, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. Crotalic crotapotins from rattlesnake venom has significantly inhibited (p<0.05) the enzymatic activity of Btae TX-I. In ex vivo experiment, Btae TX-I caused partial blockade of the neuromuscular transmission in chick biventer cervicis preparations in a similar way to other Bothrops species. Btae TX-I also inhibited contractures in the upper concentration (50 μg) to exogenous KCl (20 mM). Histological analysis of the biventer cervicis incubated with Btae TX-I showed that just the highest Btae TX-I PLA2 dose (50 μg) caused almost 27.4 ± 0.3% damaged fibers. The results give evidence that the main effect of type Asp49 Btae TX-I PLA2 from Bothriopsis taeniata is at the post-synaptic site.

Does foraging mode affect metabolic responses to feeding? A study of pygopodid lizards

Abstract Foraging mode (ambush vs. active) profoundly affects many aspects of organismal biology, including metabolic rates and their relationship with food intake. Previous studies on snakes suggest that ambushers tend to have lower standard metabolic rates (SMR) and higher energetic costs of digestion and assimilation of prey (specific dynamic action, or SDA) than do active foragers. However, phylogenetic considerations may be at least partly responsible for such patterns, as foraging mode is strongly conserved evolutionarily and most SDA studies have focused on species from only two lineages of ambush foragers (pythonid and viperid snakes) and one lineage of active foragers (colubrid snakes). We sought to deconfound the effects of phylogeny and foraging mode, investigating SMR and SDA in two closely related pygopodid lizards, the common scaly-foot Pygopus lepidopodus (active forager) and Burton’s legless lizard Lialis burtonis (ambush forager). Consistent with the pattern seen in snakes, L. burtonis exhibits a significantly lower SMR and a higher SDA than does P. lepidopodus. The magnitude of SDA in L. burtonis is comparable to that of some pythons and vipers, providing yet more evidence for the remarkable convergence between this species and ambush-foraging snakes.

Functional characterization of a slow and tight-binding inhibitor of plasmin isolated from Russell's viper venom

BackgroundSnake venoms are rich in Kunitz-type protease inhibitors that may have therapeutic applications. However, apart from trypsin or chymotrypsin inhibition, the functions of most of these inhibitors have not been elucidated. A detailed functional characterization of these inhibitors may lead to valuable drug candidates. MethodsA Kunitz-type protease inhibitor, named DrKIn-II, was tested for its ability to inhibit plasmin using various approaches such as far western blotting, kinetic analyses, fibrin plate assay and euglobulin clot lysis assay. In addition, the antifibrinolytic activity of DrKIn-II was demonstrated in vivo. ResultsDrKIn-II potently decreased the amidolytic activity of plasmin in a dose-dependent manner, with a global inhibition constant of 0.2nM. Inhibition kinetics demonstrated that the initial binding of DrKIn-II causes the enzyme to isomerize, leading to the formation of a much tighter enzyme-inhibitor complex. DrKIn-II also demonstrated antifibrinolytic activity in fibrin plate assay and significantly prolonged the lysis of the euglobulin clot. Screening of DrKIn-II against a panel of serine proteases indicated that plasmin is the preferential target of DrKIn-II. Furthermore, DrKIn-II treatment prevented the increase of FDP in coagulation-stimulated mice and significantly reduced the bleeding time in a murine tail bleeding model. ConclusionDrKIn-II is a potent, slow and tight-binding plasmin inhibitor that demonstrates antifibrinolytic activity both in vitro and in vivo. General significanceThis is the first in-depth functional characterization of a plasmin inhibitor from a viperid snake. The potent antifibrinolytic activity of DrKIn-II makes it a potential candidate for the development of novel antifibrinolytic agents.

Chorioallantoic Placentation in Viperid Snakes

Chorioallantoic Placentation in Viperid Snakes

Cold climate specialization: Adaptive covariation between metabolic rate and thermoregulation in pregnant vipers

We compared thermoregulatory strategies during pregnancy in two congeneric viperid snakes (Vipera berus and Vipera aspis) with parapatric geographic ranges. V. berus is a boreal specialist with the largest known distribution among terrestrial snakes while V. aspis is a south-European species. Despite contrasted climatic affinities, the two species displayed identical thermal preferences (Tset) in a laboratory thermal gradient. Under identical natural conditions, however, V. berus was capable of maintaining Tset for longer periods, especially when the weather was constraining. Consistent with the metabolic cold adaptation hypothesis, V. berus displayed higher standard metabolic rate at all temperatures considered. We used the thermal dependence of metabolic rate to calculate daily metabolic profiles from body temperature under natural conditions. The boreal specialist experienced higher daily metabolic rate and minimized gestation duration chiefly because of differences in the metabolic reaction norms, but also superior thermoregulatory efficiency. Under cold climates, thermal constraints should make precise thermoregulation costly. However, a shift in the metabolic reaction norm may compensate for thermal constraints and modify the cost–benefit balance of thermoregulation. Covariation between metabolic rate and thermoregulation efficiency is likely an important adaptation to cold climates.

HEAD TRIANGULATION IN TWO COLUBRINE SNAKES: PROBABLE BEHAVIOURAL REINFORCEMENT OF BATESIAN MIMICRY

ABSTRACT Two Israeli aglyphous colubrid snakes seem to mimic venomous viperid snakes. Spalerosophis diadema cliffordi resembles sympatric desert viperids, especially Vipera persica fieldi, in girth, coloration and defensive behaviour. Coluber ravergieri nummifer resembles the sympatric Vipera palaestinae in length, coloration and defensive behaviour. Direct evidence of functional mimicry is absent, except that man is frequently misled. Because several conventional conditions are fulfilled, these appear to be two cases of Batesian mimicry. Any of five hypotheses may enable the evolution of such mimicry systems in which the model may be lethal. Some individuals of both mimic species greatly increase the morphological resemblance to the models by behaviourally flattening and triangulating the head in the defensive display. This behaviour is distinct from that of flattening the head as part of a general flattening of the body, to increase the area shown an enemy or in basking. Parallely, cobras are mimicked b...