Hemorrhagic Venom Research Articles

Compendium of medically important snakes, venom activity and clinical presentations in Ghana.

Snake bite envenoming (SBE) is one neglected tropical disease that has not received the needed attention. The sequelae of burdensome disability and mortality impact the socioeconomic life of communities adversely with little documentation of SBE in health facility records in Ghana. This study details SBE and snake distribution, habits/habitats, type of venom expressed and clinical manifestations. We conducted a structured thematic desk review of peer reviewed papers, books and reports from repositories including PubMed, World Health Organization (WHO) and Women's & Children's Hospital (WCH) Clinical Toxinology Resources using bibliographic software EndNote and search engine Google Scholar with the following key words; snakes, medical importance, snake bites, venom and venom type, envenomation, symptoms and signs, vaccines, venom expenditure, strike behaviour and venom-metering + Ghana, West Africa, Africa, World. We also reviewed data from the District Health Information Management System (DHIMS) of the Ghana Health Service (GHS). Outcome variables were organized as follows: common name (s), species, habitat/habit, species-specific toxin, clinical manifestation, antivenom availability, WHO category. Snake bites and SBE were grouped by the activity of the expressed venom into neurotoxic, cardiotoxic, haemorrhagic, cytotoxic, myotoxic, nephrotoxic and procoagulants. Neurotoxic snake bites were largely due to elapids. Expressed venoms with cardiotoxic, haemorrhagic, nephrotoxic and procoagulant activities principally belonged to the family Viperidae. Snakes with venoms showing myotoxic activity were largely alien to Ghana and the West African sub-region. Venoms showing cytotoxic activity are expressed by a wide range of snakes though more prevalent among the Viperidae family. Snakes with neurotoxic and haemorrhagic venom activities are prevalent across all the agro-ecological zones in Ghana. Understanding the characteristics of snakes and their venoms is useful in the management of SBE. The distribution of snakes by their expressed venoms across the agro-ecological zones is also instructive to species identification and diagnosis of SBE.

Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species.

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A2. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions.

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of the medically most important Bitis spp. and Echis spp. African snakes.

BackgroundSnakebite envenomation exerts a heavy toll in sub-Saharan Africa. The design and production of effective polyspecific antivenoms for this region demand a better understanding of the immunological characteristics of the different venoms from the most medically important snakes, to select the most appropriate venom combinations for generating antivenoms of wide neutralizing scope. Bitis spp. and Echis spp. represent the most important viperid snake genera in Africa.Methodology/Principal findingsEight rabbit-derived monospecific antisera were raised against the venoms of four species of Bitis spp. and four species of Echis spp. The effects of immunization in the rabbits were assessed, as well as the development of antibody titers, as judged by immunochemical assays and neutralization of lethal, hemorrhagic, and in vitro coagulant effects. At the end of immunizations, local and pulmonary hemorrhage, together with slight increments in the plasma activity of creatine kinase (CK), were observed owing to the action of hemorrhagic and myotoxic venom components. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within each genus, although some antisera provided a more extensive cross-reactivity than others. The venoms that generated antisera with the broadest coverage were those of Bitis gabonica and B. rhinoceros within Bitis spp. and Echis leucogaster within Echis spp.Conclusions/SignificanceThe methodology followed in this study provides a rational basis for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against viperid venoms in sub-Saharan Africa. Results suggest that the venoms of B. gabonica, B. rhinoceros, and E. leucogaster generate antisera with the broadest cross-reactivity within their genera. These experimental results in rabbits need to be translated to large animals used in antivenom production to assess whether these predictions are reproduced in horses or sheep.

MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation

The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.

Evidence for divergent patterns of local selection driving venom variation in Mojave Rattlesnakes (Crotalus scutulatus)

Snake venoms represent an enriched system for investigating the evolutionary processes that lead to complex and dynamic trophic adaptations. It has long been hypothesized that natural selection may drive geographic variation in venom composition, yet previous studies have lacked the population genetic context to examine these patterns. We leverage range-wide sampling of Mojave Rattlesnakes (Crotalus scutulatus) and use a combination of venom, morphological, phylogenetic, population genetic, and environmental data to characterize the striking dichotomy of neurotoxic (Type A) and hemorrhagic (Type B) venoms throughout the range of this species. We find that three of the four previously identified major lineages within C. scutulatus possess a combination of Type A, Type B, and a ‘mixed’ Type A + B venom phenotypes, and that fixation of the two main venom phenotypes occurs on a more fine geographic scale than previously appreciated. We also find that Type A + B individuals occur in regions of inferred introgression, and that this mixed phenotype is comparatively rare. Our results support strong directional local selection leading to fixation of alternative venom phenotypes on a fine geographic scale, and are inconsistent with balancing selection to maintain both phenotypes within a single population. Our comparisons to biotic and abiotic factors further indicate that venom phenotype correlates with fang morphology and climatic variables. We hypothesize that links to fang morphology may be indicative of co-evolution of venom and other trophic adaptations, and that climatic variables may be linked to prey distributions and/or physiology, which in turn impose selection pressures on snake venoms.

Extremely Divergent Haplotypes in Two Toxin Gene Complexes Encode Alternative Venom Types within Rattlesnake Species

Natural selection is generally expected to favor one form of a given trait within a population. The presence of multiple functional variants of traits involved in activities such as feeding, reproduction, or the defense against predators is relatively uncommon within animal species. The genetic architecture and evolutionary mechanisms underlying the origin and maintenance of such polymorphisms are of special interest. Among rattlesnakes, several instances of the production of biochemically distinct neurotoxic or hemorrhagic venom types within the same species are known. Here, we investigated the genetic basis of this phenomenon in three species and found that neurotoxic and hemorrhagic individuals of the same species possess markedly different haplotypes at two toxin gene complexes. For example, neurotoxic and hemorrhagic Crotalus scutulatus individuals differ by 5 genes at the phospholipase A2 (PLA2) toxin gene complex and by 11 genes at the metalloproteinase (MP) gene complex. A similar set of extremely divergent haplotypes also underlies alternate venom types within C.helleri and C.horridus. We further show that the MP and PLA2 haplotypes of neurotoxic C. helleri appear to have been acquired through hybridization with C.scutulatus-a rare example of the horizontal transfer of a potentially highly adaptive suite of genes. These large structural variants appear analogous to immunity gene complexes in host-pathogen arms races and may reflect the impact of balancing selection at the PLA2 and MP complexes for predation on different prey.

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1–4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. SignificanceFull sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and compared. A novel, simplified phylogenetic tree based on the sequences of GiSPs and their closely related orthologs from other pitvipers reveals six major subtypes and offers a better understanding of vSP duplication and evolution in pitvipers of both the Old and New Worlds. It is well known that specific vSPs are potential therapeutic or diagnostic agents that target the plasma proteins or coagulation factors. Our results not only render deeper insights into the variation and evolution of vSPs, but may help to choose right venoms for the development of better therapeutic leads.

Can Surgery in Patient with Protobothrops Mucrosquamatus Envenomation be Predicted in Emergency Department?

Objective Protobothrops mucrosquamatus, a Crotalinae snake with haemorrhagic venom, is responsible for the most common poisoning snakebites in Taiwan. Although a specific antivenin has been developed to treat this snakebite, surgical intervention is still needed in some patients because of the progression of tissue injury. Early risk stratification is important to identify the early signs of need for surgery. The purpose of this study was to investigate the early predictors for surgery in patients with P. mucrosquamatus envenomation. Methods The medical records of inpatients with P. mucrosquamatus envenomation between 2008 and 2013 were retrospectively reviewed. Clinical information was collected and analysed between surgical and nonsurgical patients. Results A total of 60 patients with P. mucrosquamatus envenomation, including 8 surgical patients (13.3%) and 52 non-surgical patients (86.7%), presented to the emergency department during the study period. Compartment syndrome (62.5%) and tissue necrosis (37.5%) were the main reasons for surgery. Comparison between surgical and non-surgical patients showed significantly higher white blood cell count (17.1 × 103/μL vs. 8.5 × 103/μL; p=0.002) and peak D-dimer level (1924.8 ng/mL vs. 730.0 ng/mL; p=0.006) in the surgical group. Ecchymosis (p=0.009), haemorrhagic bulla formation (p=0.002), leukocytosis (p=0.002), elevated peak D-dimer level (>1000 ng/mL) (p=0.005), and rhabdomyolysis (creatine kinase level >1000 IU/L) (p=0.007) were the significant signs relevant to surgery. On multivariate analysis, leukocytosis and ecchymosis were the most significant predictors of surgery in patients with P. mucrosquamatus envenomation. Conclusions Patients with P. mucrosquamatus envenomation presenting with leukocytosis and wound ecchymosis will have a high probability of requiring surgical therapy. (Hong Kong j.emerg.med. 2016;23:210-219)

Unraveling the distinctive features of hemorrhagic and non-hemorrhagic snake venom metalloproteinases using molecular simulations.

Snake venom metalloproteinases are important toxins that play fundamental roles during envenomation. They share a structurally similar catalytic domain, but with diverse hemorrhagic capabilities. To understand the structural basis for this difference, we build and compare two dynamical models, one for the hemorrhagic atroxlysin-I from Bothrops atrox and the other for the non-hemorraghic leucurolysin-a from Bothrops leucurus. The analysis of the extended molecular dynamics simulations shows some changes in the local structure, flexibility and surface determinants that can contribute to explain the different hemorrhagic activity of the two enzymes. In agreement with previous results, the long Ω-loop (from residue 149 to 177) has a larger mobility in the hemorrhagic protein. In addition, we find some potentially-relevant differences at the base of the S1' pocket, what may be interesting for the structure-based design of new anti-venom agents. However, the sharpest differences in the computational models of atroxlysin-I and leucurolysin-a are observed in the surface electrostatic potential around the active site region, suggesting thus that the hemorrhagic versus non-hemorrhagic activity is probably determined by protein surface determinants.

Transcriptome and proteome of the highly neurotoxic venom of Gloydius intermedius

The venomics of Gloydius intermedius were investigated using expressed sequence tags (ESTs) analyses, 2D gel-electrophoresis combined with MALDI-TOF/TOF, and LC-MS/MS. A total of 1920 ESTs from the venom gland cDNA library were sequenced; 74% of them belonged to toxin-families. The four most abundant families among the toxin transcripts were: serine protease (SP, 36.2%), bradykinin potentiating peptide (25.3%), l-amino acid oxidase (LAAO, 13.1%), and phospholipase A2 (PLA2, 9.9%). Moreover, the full sequences of four PLA2s, eight SPs, cysteine-rich secretory protein (CRISP), C-type-lectin-like-protein (CTLP), hyaluronidase, metalloproteinase, and nerve growth factor were deduced from the cDNA sequences. Excluding the CRISP and hyaluronidase, most of the G. intermedius venom proteins bear 92–99% sequence identities to those of other pitviper venoms. The most abundant components are PLA2s (37%), SPs (20%) and LAAO (6%), while metalloproteinase, CTLP, and other components each account for <3% of the total venom proteins. The abundance of Gintexin (a crotoxin-like neurotoxin) and low levels of hemorrhagic metalloproteases, disintegrins and CTLPs highlight the great venom differences between G. intermedius and other hemorrhagic pitvipers. The bimorphism of hemorrhagic and neurotoxic venoms among Gloydius is confirmed; our results shed more lights on the co-evolution of both neurotoxicity and hypotension in some viperid venoms.

The transcriptomic and proteomic basis for the evolution of a novel venom phenotype within the Timber Rattlesnake (Crotalus horridus).

The genetics underlying adaptive trait evolution describes the intersection between the probability that particular types of mutation are beneficial and the rates they arise. Snake venoms can vary in a directly meaningful manner through coding mutations and regulatory mutations. The amounts of different components determine venom efficacy, but point mutations in coding sequences can also change efficacy and function. The Timber Rattlesnake (Crotalus horridus) has populations that have evolved neurotoxic venom from the typical hemorrhagic rattlesnake venom present throughout most of its range. We identified only a handful of nonsynonymous differences in just five loci between animals with each venom type, and these differences affected lower-abundance toxins. Expression of at least 18 loci encoding hemorrhagic toxins was severely reduced in the production of neurotoxic venom. The entire phospholipase A2 toxin family was completely replaced in the neurotoxic venom, possibly through intergeneric hybridization. Venom paedomorphosis could, at best, explain only some of the loss of expression of hemorrhagic toxins. The number of potential mechanisms for altering venom composition and the patterns observed for C.horridus suggest that rapid venom evolution should occur primarily through changes in venom composition, rather than point mutations affecting coding sequences.

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers

The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

Purification and characterization of a metalloproteinase, Porthidin-1, from the venom of Lansberg’s hog-nosed pitvipers (Porthidium lansbergii hutmanni)

Porthidium lansbergii hutmanni is a small pit viper found on Margarita Island, Venezuela. Local tissue damage is one of the most obvious characteristics of P. l. hutmanni envenomation, which can lead to diverse pathological effects, such as hemorrhage, edema, blistering, necrosis, lymphatic vessel damage and degradation of extracellular matrix. Metalloproteinases are one of the major components in venoms responsible for these effects. To date, very little is known or has been reported on P. l. hutmanni venom. Crude P. l. hutmanni venom had a LD50 of 2.5 mg/kg and was considered very hemorrhagic (minimal hemorrhagic dose [MHD]: 0.98 μg) when compared to other hemorrhagic (Bothrops) venoms in Venezuela. Crude P. l. hutmanni venom also inhibited ADP-induced platelet aggregation. A metalloproteinase, Porthidin-1, from this venom was isolated by three chromatography steps (Sephadex G100, Superose 12 HR10/30 and Bioscale Q2). Porthidin-1 falls in the SVMP P-I class having a molecular weight of 23 kDa, verified by both SDS-PAGE and mass spectrometry. High-resolution mass spectrometry and a database search identified a peptide from Porthidin-1 (YNGDLDK) belonging to the SVMP family of proteins. Porthidin-1 contained hemorrhagic, fibrino(geno)lytic, caseinolytic and gelatinolytic activities, and these activities were capable of being neutralized by metalloproteinase inhibitors but not serine proteinase inhibitors. The peptide YNGDLDK shared similarities with five venom proteins with a BLAST e-value of <1. This work details the biochemical and pathophysiological effects that can result from envenomations, and highlights the importance and significance for characterizing unknown or poorly documented venoms from different geographical regions.

毒蛇咬伤台湾经验

台湾地处亚热带，地形多高山、丛林，气候温暖潮湿，是毒蛇生长繁殖的最佳环境。其中有6种毒蛇的咬伤较为常见且常有严重的临床症状，较有流行病学上的意义，它们是赤尾鲐(Trimeresurus stejnegeri)、龟壳花(T.mucrosquamatus)、眼镜蛇(Naja naja atra)、雨伞节(Bungarus multicinctus)、百步蛇(Deinagkistrodon acutus)、锁蛇(Daboia russelli formosensis)(2)。赤尾鲐、龟壳花及百步蛇咬伤主要以出血症状为主，一般称之出血性毒蛇。眼镜蛇和雨伞节咬伤因其具神经毒性，故称之为神经性毒蛇。锁炼蛇在动物实验及临床上兼具神经及出血症状，另外归为两者兼有之毒蛇。出血性毒毒素可造成咬伤处肿胀、淤血、疼痛；严重时并可导致血凝不全、全身性出血等症状。神经性毒毒素可以作用于运动神经与肌肉接合处，而造成横纹肌不收缩，迅速导致呼吸麻痹。混合性毒毒素具有神经毒性、溶血、横纹肌溶解及肾毒性等症状。赤尾鲐的咬伤是台湾最常见的毒蛇咬伤，龟壳花次之，眼镜蛇咬伤占第三位，不过赤尾鲐咬伤有集中在台湾中部的倾向，雨伞节咬伤占第四位，锁炼蛇占第五位、百步蛇咬伤个案在六大毒蛇中反而最少，不过锁炼蛇及百步蛇咬伤主要以台湾东南部为主[4,22-23]。一般毒蛇咬伤后在4~6小时即会有症状出现，此时应尽速给予正确的抗蛇毒血清（量及种类），雨伞节咬伤尚需考虑插管使用呼吸器维持呼吸，若4~6小时内未出现症状，则最好观察6~12小时确定无症状再离院，无症状的毒蛇咬伤并不需给予抗蛇毒血清治疗，仅需一般症状治疗。蛇咬时应保持冷静，辨别蛇种及蛇的大小，并注意有无听到嘶嘶的喷气声以帮助医护人员诊断。抗蛇毒血清使用的剂量依咬伤的严重度、病人体重及咬伤后的时间长短给予不同的剂量，根据大多数毒物专家建议大人与小孩毒蛇咬伤应给予相同剂量，饭匙倩咬伤为6~12瓶、雨伞节咬伤为2~4瓶、龟壳花咬伤为2~4瓶、赤尾鲐咬伤为1~2瓶、百步蛇咬伤为2~4瓶、锁链蛇咬伤为2~4瓶，抗蛇毒血清以静脉滴注30~60分钟，毒蛇咬伤后4小时内给予效果最好，超过8小时才注射效果较差。急诊医护人员应根据毒蛇咬伤的病史，临床进展的症状及实验室的检查报告来决定适时给予何种抗蛇毒血清的注射量，滴注期间应密切注意病患各种生理现象的变化，才能降低毒蛇咬伤的死亡率。 Located at the juncture of tropical and subtropical regions, Taiwan has a warm and humid climate with abundant precipitation and food, which coupled with the island’s diverse vegetation and landscape, makes it a suitable environment for many snake species. Among these, there are six kinds of poisonous snake with epidemiological significance. Three species induce hemorrhagic symptoms (Trimeresurus mucrosquamatus, TM; Trimeresurus stejnegeri, TS; and Deinagkistrodon acutus, DA); two species induce neurotoxic symptoms (Naja naja atra, NA; and Bungarus multicinctus, BM); the other species induces hemorrhagic and neurotoxic symptoms (Vipera russelli formosensis. VR).The hemorrhagic venom causes disorders of the clotting cascade such as prolonged bleeding, primary fibrinolysis and disseminated intravascular coagulopathy. The neurotoxic venom provokes respiratory distress from weakened respiratory muscles, blurred vision, diplopia, dysarthria, dysphagia, dysphonia and paralysis of extremity muscles. The mixed envenomation manifests as a combination of neurotoxic and hemorrhagic effects previously described, as well as rhabdomyolysis and acute renal failure. The identification of snake species is important if antivenom is to be used. However, the species is not ably identified if the patient or companion fails to see or catch the responsible snake. Although these unidentifiable snakes could be non-venomous, venomous species are not uncommon. Therefore, guidelines for snakebite identification based on clinical symptoms and laboratory analysis is important to improve clinical diagnosis of snakebites. In Taiwan, T. stejnegeri bites are the most common. The second is T. mucrosquamatus. Naja naja atra is the third, Bungarus multicinctus is the forth and Vipera russelli formosen-sis is the fifth. Deinagkistrodon acutus is the least. Bites by Deinagkistrodon acutus and Vipera russelli for-mosensis generally occur in the south and east parts of the island. Aggressive antivenom treatment can reduce snakebite mortality rate, but for Bungarus multicinctus bites, maintaining the patient’s airway and supporting their ventilation is vital to reducing mortality rate in addition to antivenom treatment. For dry bites or no en-venomation, the patient should be observed for at least 6 - 12 hours before discharged. The emergency physi-cian should determine the severity of envenomation and predominate venom activity before decide what type of antivenin to administer, how much, and over what period. History of exposure, local effect and systemic syndromes of envenomation, progression of symptoms and signs, laboratory data obtained in emergency de-partment should guide the decision for antivenom therapy. The freeze-dried antivenom was diluted with 50 - 100 mL of normal saline and infused intravenously for half to one hour. The poisonous snake bites in the lat-ter 4 hours to give the effect to be best, surpasses 8 hours only then to inject the effect to be bad. The dosage most toxicologists used for treating pediatric patients with snakebites was the same as that for adults. In gen-eral, 6 - 12 vials of antivenom against neurotoxic venom were used for Naja atra bite and 2 - 4 vials for Bun-garus multicinctus; 1 - 2 vials of antivenom against hemotoxic venom was used for Trimeresurus stejnegeri and 2 - 4 vials for Trimeresurus mucrosquamatus; 2 - 4 vials of anti-Deinagkistrodon acutus were used for Deinagkistrodon acutus and 2 - 4 vials of anti-Vipera russelli formosensis were used for Vipera russelli for-mosensis bite. During the infusion, the blood pressure, consciousness and skin reaction were evaluated. Keeping the varied clinical manifestations of snake bite in mind is important for effective management. Ready availability and appropriate use of antivenin, close monitoring of patients and institution of ventilatory support, all help reduce mortality.

Immediate early inflammatory gene responses of human umbilical vein endothelial cells to hemorrhagic venom

This report describes a focused immediate early gene response by human umbilical vein endothelial cells (HUVEC) to Echis carinatus snake venom. Primary cultures of HUVEC were used to assess acute inflammatory gene responses. Crude E. carinatus venom (2.5µg/ml) was used to stimulate HUVEC. HUVEC stimulated for 3h with E. carinatus venom showed a focused response to the venom, with significant increases in metallothionein (e.g., MT1H, MT2A, MT1X) and cytochrome P450 (e.g., CYP1A1, CYP1B1) gene expressions compared to non-stimulated controls. Several other genes involved in cell growth and matrix attachment were repressed [e.g., thrombospondin 1 (THBS1), connective tissue growth factor (CTGF)]. These data suggest that acute vascular injury induced by hemorrhagic snake venom initiates an anti-oxidant response primarily involving metallothioneins.

Intra-specific variation in venom of the African Puff Adder ( Bitis arietans): Differential expression and activity of snake venom metalloproteinases (SVMPs)

Bitis arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability.

Impact of Regional Variation in Bothrops asper Snake Venom on the Design of Antivenoms: Integrating Antivenomics and Neutralization Approaches

Intraspecific snake venom variations have implications in the preparation of venom pools for the generation of antivenoms. The impact of such variation in the cross-reactivity of antivenoms against Bothrops asper venom was assessed by comparing two commercial and four experimental antivenoms. All antivenoms showed similar immunorecognition pattern toward the venoms from adult and neonate specimens. They completely immunodepleted most P-III snake venom metalloproteinases (SVMPs), l-amino acid oxidases, serine proteinases, DC fragments, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like proteins, and partially immunodepleted medium-sized disintegrins, phospholipases A(2) (PLA(2)s), some serine proteinases, and P-I SVMPs. Although all antivenoms abrogated the lethal, hemorrhagic, coagulant, proteinase, and PLA(2) venoms activities, monospecific experimental antivenoms were more effective than the polyspecific experimental antivenom. In addition, the commercial antivenoms, produced in horses subjected to repeated immunization cycles, showed higher neutralization than experimental polyspecific antivenom, produced by a single round of immunization. Overall, a conspicuous pattern of cross-neutralization was evident for all effects by all antivenoms, and monospecific antivenoms raised against venom from the Caribbean population were effective against venom from the Pacific population, indicating that geographic variations in venom proteomes of B. asper from Costa Rica do not result in overt variations in immunological cross-reactivity between antivenoms.

Snake Venomics of the Central American Rattlesnake Crotalus simus and the South American Crotalus durissus Complex Points to Neurotoxicity as an Adaptive Paedomorphic Trend along Crotalus Dispersal in South America

We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A(2) (PLA(2) crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn(2+)-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD(50)), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North-South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased concentration of the neurotoxins crotoxin and crotamine in South American rattlesnake venoms strongly argues that the gain of neurotoxicity and lethal venom activities to mammals may have represented the key axis along which overall venom toxicity has evolved during Crotalus durissus invasion of South America. The paedomorphic trend is supported by a decreasing LNC (lethal neurotoxicity coefficient, defined as the ratio between the average LD(50) of the venom and the crotoxin + crotamine concentration) along the North-South axis, coincident with the evolutionary dispersal pattern of the Neotropical rattlesnakes. The indistinguisable immunoreactivity patterns of Costa Rican and Venezuelan polyvalent antivenoms toward C. simus and C. durissus venoms strongly suggest the possibility of using these antivenoms indistinctly for the management of snakebites by adult C. simus and by certain C. d. cumanensis populations exhibiting a hemorrhagic venom phenotype. The antivenomic results also explain why the antivenoms effectively neutralize the hemorrhagic activity of adult C. simus venoms but does not protect against adult C. durissus sp. and newborn C. simus envenomations. The identification of evolutionary trends among tropical Crotalus, as reported here, may have an impact in defining the mixture of venoms for immunization to produce an effective pan-American anti-Crotalus antivenom.

Molecular characterisation of endogenous snake venom metalloproteinase inhibitors

Viper venoms contain one of the most potent mixtures of proteases in natural existence and yet the venom gland and proteins in this mixture are refractory to degradation. Here we demonstrate that the sub-10-kDa components of venom from two African viper species ( Echis ocellatus and Cerastes cerastes cerastes) are predominantly composed of the tri-peptide pyroglutamate-lysine-tryptophan (pEKW). This tripeptide is encoded by tandemly repeating elements and, in E. ocellatus, on the same transcript as a novel, and highly unusual, poly-histidine-poly-glycine peptide (pHpG) also detected in E. ocellatus venom. The pEKW and pHpG peptides inhibit the proteolytic activity of the haemorrhagic snake venom metalloproteinase (SVMP), EoVMP-2, and the haemorrhagic activity of E. ocellatus venom. These results demonstrate that these vipers express abundant transcripts encoding tandemly repeated protease inhibitor cassettes and accumulate significant quantities of peptide inhibitors in venoms to provide a basis for attenuating the proteolytic activity of SVMPs.

Comparative analysis of the catalytic domain of hemorrhagic and non-hemorrhagic snake venom metallopeptidases using bioinformatic tools

Snake venom metalloproteases (SVMPs) are a set of interesting enzymes that are one of the major components of venom affecting hemostasis. A great challenge since their discovery has been to find molecular features responsible for their hemorrhagic potency and many attempts have been made without any consistent result. Here we describe a series of comparisons between the catalytic domains of hemorrhagic and non-hemorrhagic SVMPs made with the help of bioinformatics. These involved sequence and structure-based multiple alignments, phylogenetic reconstruction, predicted physical and chemical properties, motif scanning and structural analyses. Although hemorrhagic activity seems to be complex, involving multiple factors, we found some molecular characteristics that may influence the toxic effects. Among these findings, it was possible to use a molecular surface feature to subdivide the P-I class in hemorrhagic and non-hemorrhagic SVMPs. It was also possible to suggest a role for the conserved Asp148 and Ser176 residues in the stabilization of the active site.