Vipers of Major clinical relevance in Europe: Taxonomy, venom composition, toxicology and clinical management of human bites.

Snakebites in Europe are mostly due to bites from Viperidae species of the genus Vipera. This represents a neglected public health hazard with poorly defined incidence, morbidity and mortality. In Europe, fourteen species of "true vipers" (subfamily Viperinae) are present, eleven of which belong to the genus Vipera. Amongst these, the main medically relevant species due to their greater diffusion across Europe and the highest number of registered snakebites are six, namely: Vipera ammodytes, V. aspis, V. berus, V. latastei, V. seoanei and V. ursinii. Generally speaking, viper venom composition is characterised by many different toxin families, like phospholipases A2, snake venom serine proteases, snake venom metalloproteases, cysteine-rich secretory proteins, C-type lectins, disintegrins, haemorrhagic factors and coagulation inhibitors. A suspected snakebite is often associated with severe pain, erythema, oedema and, subsequently, the onset of an ecchymotic area around one or two visible fang marks. In the field, the affected limb should be immobilised and mildly compressed with a bandage, which can then be removed once the patient is being treated in hospital. The clinician should advise the patient to remain calm to reduce blood circulation and, therefore, decrease the spread of the toxins. In the case of pain, an analgesic therapy can be administered, the affected area can be treated with hydrogen peroxide or clean water. However, anti-inflammatory drugs and disinfection with alcohol or alcoholic substances should be avoided. For each patient, clinical chemistry and ECG are always a pre-requisite as well as the evaluation of the tetanus immunisation status and for which immunisation may be provided if needed. The treatment of any clinical complication, due to the envenomation, does not differ from treatments of emergency nature. Antivenom is recommended when signs of systemic envenomation exist or in case of advanced local or systemic progressive symptoms. Recommendations for future work concludes. The aim of this review is to support clinicians for the clinical management of viper envenomation, through taxonomic keys for main species identification, description of venom composition and mode of action of known toxins and provide a standardised clinical protocol and antivenom administration.

Insights into the Evolution of a Snake Venom Multi-Gene Family from the Genomic Organization of Echis ocellatus SVMP Genes.

The molecular events underlying the evolution of the Snake Venom Metalloproteinase (SVMP) family from an A Disintegrin And Metalloproteinase (ADAM) ancestor remain poorly understood. Comparative genomics may provide decisive information to reconstruct the evolutionary history of this multi-locus toxin family. Here, we report the genomic organization of Echis ocellatus genes encoding SVMPs from the PII and PI classes. Comparisons between them and between these genes and the genomic structures of Anolis carolinensis ADAM28 and E. ocellatus PIII-SVMP EOC00089 suggest that insertions and deletions of intronic regions played key roles along the evolutionary pathway that shaped the current diversity within the multi-locus SVMP gene family. In particular, our data suggest that emergence of EOC00028-like PI-SVMP from an ancestral PII(e/d)-type SVMP involved splicing site mutations that abolished both the 3' splice AG acceptor site of intron 12* and the 5' splice GT donor site of intron 13*, and resulted in the intronization of exon 13* and the consequent destruction of the structural integrity of the PII-SVMP characteristic disintegrin domain.

Comparative Studies of Structural and Functional Properties of Snake Venom Metalloproteinases.

Snake venom metalloproteinases (SVMPs) induces local and systemic effects on patients suffering from snakebite, degrading extracellular matrix (ECM) proteins such as collagen, gelatin, elastin, laminin, fibronectin, nidogen (entactin), and thrombospondin that cause local hemorrhage and tissue damage. They cleave or activate coagulation factors such as fibrinogen, fibrin, prothrombin, factor V, factor IX, factor X and protein C that bring about systemic coagulopathy. SVMPs and their truncated forms cleave or interfere with platelet adhesive proteins such as vWF, fibrinogen and collagen, and cleave or interfere with platelet receptors such as GPVI, alpha2beta1, GPIb, GPIX, and GPIIbIIIa that result in platelet aggregation defect. SVMPs induce cancer cell line to form morphological changes and apoptosis in vitro concordant with skin necrosis after snakebite in some cases. These local effects caused by SVMPs have no certain treatments, even with commercial anti-venom. SVMPs researches are focusing on their inhibitors, measurement and replacement of blood coagulation factor defects, or anti-cancer drug.

Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: a study of paraspecificity.

We conducted an extensive study of neutralization of lethality of 11 species and one subspecies of snakes of the genus Vipera, and of five species of Macrovipera, by two experimental equine antisera. One antiserum was a trivalent preparation raised against the venoms of Vipera aspis aspis, Vipera berus berus and Vipera ammodytes ammodytes; the other was a pentavalent preparation that also included venoms of Vipera (now Montivipera) xanthina and Macrovipera lebetina obtusa. We measured specific neutralization of lethality against all venoms included in the immunization schemes, and paraspecific neutralization against the venoms of Vipera ammodytes montandoni, Vipera (Montivipera) bornmuelleri, Vipera latastei, Vipera (Mo.) latifii, Vipera (Mo.) lotievi, Vipera (Daboia) palaestinae, Vipera (Mo.) raddei and Vipera seoanei, as well as against Macrovipera (D.) deserti, Macrovipera lebetina cernovi, Macrovipera lebetina turanica and Macrovipera schweitzeri. We found an important degree of paraspecific protection within each genera (omitting recent reclassification) that was quite independent of both the lethal potency of the venoms and their geographic origin. This information may be of use to clinicians charged with the treatment of Vipera or Macrovipera envenomations with non-specific antivenoms.

Snakebites in Hungary--epidemiological and clinical aspects over the past 36 years.

Epidemiological and clinical aspects of snakebites in Hungary between 1970 and 2006 were surveyed. A total of 97 cases were recorded from 21 species, including the two native vipers, Vipera berus and Vipera ursinii, and various exotic species represented by Viperidae, Elapidae, and Colubridae. Bites by native species on laymen are uncommon (17 cases) and present trivial clinical manifestations. Compared with the consequences of native Vipera cases, bites by exotic species often resulted in severe or life-threatening envenomations. These cases were treated with antivenom administration, plasmapheresis, fasciotomy, and amputation. There were two fatalities caused by V. berus and Agkistrodon contortrix. Both of these cases were inflicted in snake-handlers with a previous history of Viperidae bites and the cause of deaths are attributed to anaphylactic reactions as a consequence of hypersensitivity to the venom. Snake-handlers and their physicians face a major challenge due to the diversity and severity of signs and symptoms following exotic venomous snakebites, and the risk of anaphylaxis or anaphylactoid reactions in patients with repeated exposure to snake venom and antivenom. Highly dangerous venomous snake species continue to appear in collections of Hungarian snake-handlers.

High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions.

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21 (DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 degrees C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg2+ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.

An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins.

Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124-->I/K128-->E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128-->E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108-->N/K111-->N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128-->E) is equivalent to the AtxC(I124-->F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.

Biological activities of [Thr2]sarafotoxin-b, a synthetic analogue of sarafotoxin-b.

The 21 amino acid sarafotoxins (SRTX) c and d/e as well as endothelin-3 (ET-3) are known to be less toxic and weaker pharmacologically than the other isopeptides SRTX-a, SRTX-b and ET-1. Since SRTX-c, SRTX-d/e and ET-3 possess a Thr instead of a Ser at position 2, we investigated the possibility that this mutation could be responsible for the observed biological differences. Here we show that the synthetic [Thr2]SRTX-b has indeed a lower vasoconstriction efficacy (approximately 35%) in the rabbit aorta, but it is nearly as potent as SRTX-b in toxicity tests and in influencing contraction of the rat uterus. Using monoclonal antibodies directed against the structurally related endothelin-1, we also show that the antigenicity of the analogue is comparable to that of SRTX-b, suggesting that the overall structure of the two peptides is similar, despite the substitution at position 2. We suggest that the Thr2 substitution contributes to the lower activity of the 'weak' peptides in some systems; however, additional substitutions found in the 'weak' peptides of the ET/SRTX family most probably contribute to their low pharmacological activity.

A comparative study on the electrophoretic patterns of snake venoms.

1. Examination of the polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns of snake venoms shows that these patterns are useful for species differentiation (and hence identification) for snakes of certain genera but have only limited application for snakes from some other genera, due either to the marked individual variations in the venoms or the lack of marked interspecific differences within the same genus. 2. There is no substantial intersubspecific difference in the electrophoretic patterns of the venoms. 3. In general there are no common characteristics in the electrophoretic patterns of the venom at the generic level because of the wide variations in the electrophoretic patterns of venoms of snakes within the same genus. 4. At the familial level, the venoms of Elapidae exhibited SDS-PAGE patterns distinct from those of Crotalidae.