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The saw-scaled viper (or carpet viper) is endemic to Asia and often found in the Indian subcontinent. Twelve species of Echis are found worldwide, though Echis carinatus carinatus (the Indian saw-scaled viper) is found in peninsular India and it has occupied a key position in the list of category I medically important snakes in India. E. carinatus can grow to be 38–80 cm in length; its color varies from grayish to reddish, olive, or pale brown ground color; and its most common pattern is whitish spots with dark-brown edges. It is distributed in the Indian subcontinent, except in eastern and northeastern regions of the country, Gangetic plains, and Himalayan foothills. On average, the yield of milked venom from an adult saw-scaled viper is approximately 12 mg, but the lethal dose for an adult person is estimated to be only 5 mg. Due to lack of a comprehensive study, the geographical variation in venom composition in E. carinatus across Indian states has not been established. The venom proteome composition of E. c. carinatus from southern India has been analyzed by tandem mass spectrometry and 90 distinct toxins have been identified (enzymatic and nonenzymatic toxin classes) from 15 snake venom protein families. The relative abundance of different toxins in E. carinatus venom is presented in this chapter. The E. c. carinatus venom is hemotoxic, but in comparison to Russell’s viper venom, Echis envenomation has a more pronounced effect on blood coagulation. Acute renal failure, bleeding, and hypotension following E. carinatus envenomation are frequently reported toxic effects of the venom.

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.

INTRODUCTION: Two venomous snakes, the asp viper (Vipera aspis) and the common adder (Vipera berus) are native to Switzerland. Bites by both vipers cause mainly local effects, but systemic envenomation is possible. METHODS: We analysed all calls concerning indigenous venomous snakebites recorded at the Swiss National Poisons Information Centre between 1997 and 2018, including all cases with identification by a herpetologist, and/or with compatible symptoms and circumstances of the exposure. RESULTS: During the study period, 1,364 cases concerned snakebites. One third (466; 34%) were attributed to indigenous vipers. In 243 (52%) patients, medical follow-up information was available, with good causality between exposure and symptoms in 219 (90%) patients. Vipera aspis was identified in 77 of the cases (35%), Vipera berus in 54 (25%), and not further specified vipers in 88 (40%). In over two thirds of the 219 cases (155, 71%) adult patients were affected (male 109, female 46; median age 43 years [range 16-90]). Sixty-four patients were children (male 47, female 16; median age 11 years [range 1.3-15.9]). The highest occurrence of bites was in the summer months. In the majority of patients, the clinical course was mild (94; 43%) or moderate (80; 36%); a lower proportion was either asymptomatic (17; 8%) or exhibited severe symptoms (28; 13%). There were no fatalities reported. The most frequent symptoms were local effects at the bite site with mild (100; 46%) to moderate (56; 25%) swelling, pain (65; 30%) and redness (51; 23%). Gastrointestinal symptoms including nausea (31; 14%), vomiting (47; 22%) and abdominal pain (25; 11%) were also common. Other systemic symptoms included cardiovascular effects (e.g., hypotension (20; 9%) or shock [6; 3%]), neurotoxicity (e.g., visual impairment [5; 2.3%]) and haematotoxicity (e.g., coagulopathy [11; 5%]). Seven (3.2%) patients developed anaphylactic reactions. Antivenom was administered in only 20% (24 with moderate and 19 with severe symptoms) with good resolution of symptoms. The mean duration of hospitalization was 2 days (0-12 days). CONCLUSION: Snakebites in Switzerland can result in severe symptoms, sometimes necessitating antivenom treatment., Swiss Medical Weekly, 151, ISSN:1424-7860, ISSN:1424-3997

76 páginas, 2 tablas, 8 figuras, 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.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite., Snakebite is a life-threatening neglected tropical disease that is currently treated using different antibody-based antivenoms, each effective against bites of specific snake species, but not others. Here, the authors show that a combination of two toxin-inhibiting repurposed drugs provides broad protection in experimental animals against the lethal effects of snakebites from multiple snake species.

Venom is a functional adaptation that has evolved independently in almost every major animal lineage for use in defence, predation, and/or competition. The complexity and diversity of components formulating the venom mixture has resulted in a phenomenon whereby the composition of a species’ venom can vary down to the level of the individual. Venom variation produces an undesirable outcome from an anthropogenic perspective as the efficacy of an antivenom may be compromised due to variation in the antigenicity of venoms. This complicates the research efforts that seek to ameliorate the global impact of snakebite. The Viperinae subfamily of vipers contains some of the most medically significant snakes world-wide, often responsible for majority proportions of snakebite in the regions they inhabit. Despite their medical importance, Viperinae is an understudied clade. There is a need to characterise the bioactivity, composition, and variation of venom in these snakes in order to develop a deeper understanding of the complex interactions between snake ecology, evolution, venom, and snakebite envenoming. Accordingly, the aim of this work is threefold: 1. To characterise the venom activity of select viperine genera that are underrepresented in the existing literature, focussing on venom variation; 2. To explore links in the evolutionary relationships between snake ecology and venom biochemistry; and 3. To investigate the effectiveness of antivenoms relative to venom activity and variability. The thesis begins with a broad overview of the evolutionary history, biology, and ecology of the viperines and their venom in Chapter 1. Chapter 2 contains study protocols. Chapter 3, which comprises three sub-chapters, focusses on the genus Daboia. It begins with a characterisation of the proteolytic and haemotoxic properties of venoms from twelve geographical variants of five Daboia species, followed by an assessment of antivenom cross-reactivity, and finishes with a critical comparative analysis of the limitations and advantages offered by a suite of commonly used antivenom assessment protocols. Chapter 4 consists of two sub-chapters focussing on the neglected genera Pseudocerastes and Eristicophis. The first section of the chapter entails a comprehensive analysis of the neurotoxic, haemotoxic, and cytotoxic bioactivities of the venoms, and the second section evaluates antivenom cross-reactivity for the clade. The thesis is concluded in chapter 5.These research aims were achieved by adopting an integrated proteomic, bioactivity, and immunogenic assay approach, and the venoms of Daboia, Pseudocerastes, and Eristicophis were evaluated using all or a combination of the following methods: Venom protein composition was assessed via SDS-PAGE gel analysis. Generalised proteolytic activity, factor X and prothrombin activation, and total thrombin generation by the venoms were assessed via kinetic fluorescence assays. Coagulant activity of venoms on human plasma was quantified using an automated coagulation analyser. Venom-induced kinetics and structure of human, amphibian, and avian plasma clots were assessed via thromboelastography. Activity of venoms on fibrinogen was evaluated by thromboelastography and imaged via fibrinogen gels. Interactions of the venoms with phospholipid, tissue factor, and plasminogen activator were testing using chromogenic assays. Neurotoxicity testing was conducted via chick biventer assays. Cytopathic activity testing was conducted on PaTu-T, NFF, and MM96L cells, quantified via MTT assays, and visualised via light microscopy. Antivenom effectiveness was assessed functionally by using an automated coagulation analyser to measure shifts in coagulation time, as well as immunogenically via western blotting and ELISAs (endpoint titration and chaotropic). The advantages and limitations of a range of antivenom assessment assays were also evaluated.The previously unknown venom activities of Pseudocerastes urarachnoides were revealed to exert strong procoagulant activity, activating both prothrombin and factor X. Most other species tested also possessed strong, procoagulant venoms, with exception of the Afro-Arabian species of Daboia (D. palaestinae and D. deserti) and two species of Pseudocerastes (P. persicus and P. fieldi). Fibrinogenolytic activity across the clade was extremely diverse. Intragenus and intraspecies variation in venom activity was extreme throughout all assays. The variability of these venoms typically had a detrimental impact on the effectiveness of antivenoms, whereby many antivenoms proved inadequate. Their neutralisation of venom activities was inconsistent and ranged from good coverage for some species’ venoms to no coverage for others. These studies present the first comprehensive functional analyses of Pseudocerastes and Eristicophis venoms, representing a key contribution to our knowledge of viper venom composition and activity. Crucially, antivenom effectiveness and cross-reactivity for this clade has been described for the first time. In addition, this work has given greater depth and breadth to the scope of research that exists surrounding the geographic variation in venom composition of Daboia and the impact this has on antivenom effectiveness. This will aid in the treatment of envenomings by thesespecies, and thus positively contributes to the global efforts seeking to address snakebite mortality and morbidity. This series of studies provides a critical data set which informs antivenom manufacturers, clinicians, and evolutionary toxinologists in the field of venom research.

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV, The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T.albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

Gloydius caucasicus (NIKOLSKY, 1916) is a member of the Viperidae family in Iran. Comprehensive understanding of the toxigenic characteristics of snake venom is important for clinical monitoring of snakebite patients and effective therapy. We compared the toxic activities of venoms and the neutralization capacity of antivenoms produced with venoms from wild adult (WA) with long-term captive adult (LCA) of G. caucasicus in order to obtain more effective antivenom from LCA in therapy, and subsequently protect G. caucasicus from overharvesting for its venom, which poses a real threat of extinction for the species. Our results showed that LD50 of WA and LCA were 16.8 μg/dose and 17.7 μg/dose, respectively. Lower hemorrhagic and necrotic (p ≥ 0.05), and higher coagulative and edematogenic activities (p ≤ 0.05) were observed in WA compared with LCA venom. Also, captive-born neonates exhibited weaker toxic activities compared with captive adult snakes, which could be an age-related difference. Study data illustrated that effective capacity of LCA antivenom to neutralize the toxic activities of WA viper venom. According to the results, about 0.4–4 μl of LCA antivenom is required to neutralize the toxic activities of 1 μg of WA venom, indicating its efficacy in treatment of snakebites in humans. On this basis, it is recommended that capture of wild snakes for their venom be discontinued to reduce their future extinction risk.

Physicians think of mast cells and IgE primarily in the context of allergic disorders, including fatal anaphylaxis. This 'bad side' of mast cells and IgE is so well accepted that it can be difficult to think of them in other contexts, particularly those in which they may have beneficial functions. However, there is evidence that mast cells and IgE, as well as basophils (circulating granulocytes whose functions partially overlap with those of mast cells), can contribute to host defense as components of adaptive type 2 immune responses to helminths, ticks and certain other parasites. Accordingly, allergies often are conceptualized as "misdirected" type 2 immune responses, in which IgE antibodies are produced against any of a diverse group of apparently harmless antigens, and against components of animal venoms. Indeed, certain unfortunate patients who have become sensitized to venoms develop severe IgE-associated allergic reactions, including fatal anaphylaxis, upon subsequent venom exposure. In this review, we will describe evidence that mast cells can enhance innate resistance, and survival, to challenge with reptile or arthropod venoms during a first exposure to such venoms. We also will discuss findings indicating that, in mice surviving an initial encounter with venom, acquired type 2 immune responses, IgE antibodies, the high affinity IgE receptor (FceRI), and mast cells can contribute to acquired resistance to the lethal effects of both honeybee venom and Russell's viper venom. These findings support the hypothesis that mast cells and IgE can help protect the host against venoms and perhaps other noxious substances.

Echis carinatus is one of the highly venomous snakes of Pakistan that is responsible for numerous cases of envenomation and deaths. In Pakistan, medicinal plants are commonly used traditionally for snakebite treatment because of their low cost and easy availability in comparison with antivenom. The current research is aimed at evaluating the inhibitory activity of Pakistani medicinal plants against acetylcholinesterase and 5′-nucleotidases present in Echis carinatus venom. Acetylcholinesterase and 5′-nucleotidase enzymatic assays were performed at different venom concentrations to check the activity of these enzymes. Methanolic extracts from different parts of plants were used for in vitro determination of their inhibitory activity against 5′-nucleotidases in snake venom. Active methanolic extracts were subsequently fractioned using different solvents, and these fractions were also assessed for their anti-5′-nucleotidase activity. Results of this study exhibited that Eugenia jambolana Willd. ex O. Berg, Rubia cordifolia L., Trichodesma indicum (L.) R. Br., Calotropis procera (Wild.) R. Br., Curcuma longa L., and Fagonia arabica L. were able to significantly (p > 0.5) neutralize the 5′-nucleotidase activity by 88%, 86%, 86%, 85%, 83.7%, and 83%, respectively, compared with a standard antidote (snake venom antiserum). Thus, this study indicates that these plants possess the potential to neutralize one of the toxic enzymatic components of Echis carinatus venom and hence can help to augment the future efforts of developing alternative therapy for the management of snakebites.

Elucidating electrokinetic stability by which surface charges regulate toxins interaction with erythrocytes is crucial for understanding the cell functionality. Electrokinetic properties of human erythrocytes upon treatment of Vipoxin, phospholipase A2 (PLA2) and Vipoxin acidic component (VAC), isolated from Vipera ammodytes meridionalis venom were studied using particle microelectrophoresis. PLA2 and Vipoxin treatments alter the osmotic fragility of erythrocyte membranes. The increased stability of cells upon viper toxins is presented by the increased zeta potential of erythrocytes before sedimentation of cells during electric field applied preventing the aggregation of cells. Lipid peroxidation of low dose toxin-treated erythrocytes shows reduced LP products compared to untreated cells. The apparent proton efflux and conductivity assays are performed and the effectiveness PLA2 > Vipoxin>VAC is discussed. The reported results open perspectives to a further investigation of the electrokinetic properties of the membrane after viper toxins treatment to shed light on the molecular mechanisms driving the mechanisms of inflammation and neurodegenerative diseases

Funding Information: This research was funded by a 7th Framework Program, SP3 Support for training and career development of researchers Marie Curie Actions (Project: PIEF-GA-2009-253014) and received support from a BBSRC-SFI research grant (BB/P001432/1) between the Applied Mycology Group at Cranfield University and the School of Biology and Environmental Science, University College Dublin, Ireland. The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms. publishersversion published

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

For most antivenoms there is little information from clinical studies to infer the relationship between dose and efficacy or dose and toxicity. Antivenom dose-finding studies usually recruit too few patients (e.g. fewer than 20) relative to clinically significant event rates (e.g. 5%). Model based adaptive dose-finding studies make efficient use of accrued patient data by using information across dosing levels, and converge rapidly to the contextually defined ‘optimal dose’. Adequate sample sizes for adaptive dose-finding trials can be determined by simulation. We propose a model based, Bayesian phase 2 type, adaptive clinical trial design for the characterisation of optimal initial antivenom doses in contexts where both efficacy and toxicity are measured as binary endpoints. This design is illustrated in the context of dose-finding for Daboia siamensis (Eastern Russell’s viper) envenoming in Myanmar. The design formalises the optimal initial dose of antivenom as the dose closest to that giving a pre-specified desired efficacy, but resulting in less than a pre-specified maximum toxicity. For Daboia siamensis envenoming, efficacy is defined as the restoration of blood coagulability within six hours, and toxicity is defined as anaphylaxis. Comprehensive simulation studies compared the expected behaviour of the model based design to a simpler rule based design (a modified ‘3+3’ design). The model based design can identify an optimal dose after fewer patients relative to the rule based design. Open source code for the simulations is made available in order to determine adequate sample sizes for future adaptive snakebite trials. Antivenom dose-finding trials would benefit from using standard model based adaptive designs. Dose-finding trials where rare events (e.g. 5% occurrence) are of clinical importance necessitate larger sample sizes than current practice. We will apply the model based design to determine a safe and efficacious dose for a novel lyophilised antivenom to treat Daboia siamensis envenoming in Myanmar., Author summary Snakebite envenoming is one of the most neglected tropical diseases considering its burden of mortality and morbidity. Antivenoms are the only known effective treatment for snake-bite envenoming but are frequently responsible for high rates of adverse reactions. Clinical development of antivenoms rarely follows the iterative phases of clinical development applied to other drugs. Dosing is typically based on pre-clinical testing. Here we propose a Bayesian model based adaptive design for phase 2 clinical trials aiming to determine the optimal dose of antivenom needed for treatment of snakebite envenoming. Optimality is defined using safety and efficacy thresholds contextual to the study. This design can be applied to all antivenoms which have binary efficacy and toxicity endpoints. Our design formally specifies a desired efficacy and a maximum tolerated toxicity. We use simulation studies to characterise adequate sample sizes to determine an approximately optimal dose under different scenarios. The simulation studies highlight the advantages of a model based design over simpler rule based alternatives. This design will be used to determine an effective and safe dose of the new lyophilised viper antivenom currently in use to treat Russell’s viper envenoming in Myanmar.

Snakebite is one of the most neglected diseases of developing countries. Deaths due to snakebite envenoming are quite high in Pakistan, and many deaths are caused by Echis carinatus envenomation. Traditional use of medicinal plants against snakebites is a common practice in Pakistan due to countless benefits. The current study was performed with the objective to evaluate eighteen Pakistani medicinal plants inhibitory potential against hyaluronidase and alkaline phosphatase enzymes of Pakistani Echis carinatus venom. Hyaluronidase activity (0.2-1.6 mg/0.1 mL) and alkaline phosphatase activity (0.1-0.8 mg/0.1 mL) were measured in dose-dependent manner. Crude methanolic extracts of medicinal plants were used for in vitro investigation of their inhibitory activity against toxic enzymes. All active plants were fractioned using different solvents and were again analyzed for inhibitory activity of same enzymes. Results indicated all plants were able to neutralize hyaluronidase that Swertia chirayita (Roxb. ex Flem.) Karst., Terminalia arjuna Wight and Arn, Rubia cordifolia Thumb., and Matthiola incana (L.) R.Br. inhibited maximum hyaluronidase activity equivalent to standard reference (p > 0.5). Pakistani medicinal plants are dense with natural neutralizing metabolites and other active phytochemicals which could inhibit hyaluronidase activity of Pakistani Echis carinatus venom. Further advanced studies at molecular level could lead us to an alternative for envenoming of Pakistani Echis carinatus venom.

Sterile inflammation (SI) is an essential process in response to snakebite and injury. The venom induced pathophysiological response to sterile inflammation results into many harmful and deleterious effects that ultimately leads to death. The available treatment for snakebite is antiserum which does not provide enough protection against venom-induced pathophysiological changes like haemorrhage, necrosis, nephrotoxicity and often develop hypersensitive reactions. In order to overcome these hindrances, scientists around the globe are searching for an alternative therapy to provide better treatment to the snake envenomation patients. In the present study TiO2 (Titanium dioxide)-NPs (Nanoparticles) has been assessed for antisnake venom activity and its potential to be used as an antidote. In this study, the synthesis of TiO2-NPs arrays has been demonstrated on p-type Silicon Si substrate (∼30 ohm-cm) and the surface topography has been detected by Field-emission scanning electron microscopy (FESEM). The TiO2-NPs successfully neutralized the Daboia russelii venom (DRV) and Naja kaouthia venom (NKV)-induced lethal activity. Viper venom induced haemorrhagic, coagulant and anticoagulant activities were effectively neutralized both in in-vitro and in vivo studies. The cobra and viper venoms-induced sterile inflammatory molecules (IL-6, HMGB1, HSP70, HSP90, S100B and vWF) were effectively neutralised by the TiO2-NPs in experimental animals.

We characterized the hemorrhagic, coagulant and defibrinogenant activities of Lachesis muta venom and evaluated the capacity of the Brazilian antivenoms in neutralizing these activities. The hemorrhagic activity of L. muta venom was similarly neutralized by Bothrops, Bothrops-Lachesis and Bothrops-Crotalus antivenoms. The coagulant and defibrinogenant activities were better neutralized by the Bothrops-Lachesis antivenom. Bothrops-Crotalus antivenom also neutralized these activities, indicating that it can be an alternative to treat Lachesis envenomations when Bothrops-Lachesis antivenom is unavailable.

Snake venoms have components with diverse biological actions that are extensively studied to identify elements that may be useful in biomedical sciences. In the field of autoimmunity and rheumatology, various findings useful for the study of diseases and potential drug development have been reported. The study of disintegrins, proteins that block the action of integrins, has been useful for the development of antiplatelet agents and principles for the development of immunosuppressants and antineoplastics. Several proteins in snake venoms act on the coagulation cascade, activating factors that have allowed the development of tests for the study of coagulation, including Russell’s viper venom time, which is useful in the diagnosis of antiphospholipid syndrome. Neurotoxins with either pre- or postsynaptic effects have been used to study neurogenic synapses and neuromuscular plaques and the development of analgesics, muscle relaxants and drugs for neurodegenerative diseases. Various components act by inhibiting cells and proteins of the immune system, which will allow the development of anti-inflammatory and immunosuppressive drugs. This review summarizes the usefulness of the components of snake venoms in the fields of autoimmunity and rheumatology, which can serve as a basis for diverse translational research., Highlights • Snake venoms possess multiple biological functions useful in translational research. • Snake venoms have various biological effects on the immune system. • Many answers about autoimmunity can be solved based on the study of snake venoms.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine- rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

We report a structural and functional proteomics characterization of venoms of the two subspecies (

Snakebites occur worldwide with varying frequency. Outcomes associated with snakebite are varied and depend on the type of snake and the general health of the patient. We report a case of snakebite by Cerastes cerastes or desert horned viper. Consumptive coagulopathy and cardiotoxicity with electrocardiographic changes complicated the clinical course of the patient. To the best of our knowledge, this is the first reported case in the state of Qatar.

The excessive production of inflammatory mediators results in an overactive immune response leading to the worsening of various human diseases. Thus, there is a still need to identify molecules able to regulate the inflammatory response. Lebecetin, a C-type lectin protein isolated from Macrovipera lebetina snake venom, was previously characterized as a platelet aggregation inhibitor and antitumor active biomolecule. In the present work, we investigated its effect on the production of some cytokines linked to inflammatory response and the underlying mechanisms in lipopolysaccharide (LPS)-induced THP1 macrophages. Interestingly, we found that lebecetin reduced the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8 while it partially increased LPS-induced secretion of the immunomodulatory cytokine IL-10. Furthermore, this modulatory effect was accompanied by decreased activation of ERK1/2, p38, AKT kinases and NF-κB along with reduced expression of αvβ3 integrin. Thus, this study highlights the promising role of lebecetin as a natural biomolecule that could manage the inflammatory response involved in the development and progression of inflammatory diseases.

Ethnopharmacological relevance Snake bite is a major occupational hazard in tropical and subtropical countries including India as per the World Health Organization. Naja naja (Indian cobra) and Daboia russelli (Russell's viper) are the two poisonous snakes commonly associated with human mortality in India. Andrographis serpyllifolia (Rottler ex Vahl) Wight has been documented in ethnobotanical records as a plant possessing potent anti-snake venom activity. Aim of the study The present study is aimed for systematic evaluation of in vitro anti-venom potential of various solvent based leaf extracts of A. serpyllifolia against toxic venom enzymes of Naja naja and Daboia russelli. Materials and methods Different solvent based leaf extracts of A. serpyllifolia were tested against the snake venoms of Naja naja and Daboia russelli obtained from Irula Snake Catchers Industrial Co-operative Society Limited, Kancheepuram, Tamil nadu, India. Three different in vitro neutralization assays such as indirect hemolysis, procoagulent and lytic activities and seven in vitro enzyme inhibition assays such as protease, acetylcholinesterase, phosphomonoesterase, phosphodiesterase, 5′nucleotidase, phospholipase A2, hyaluronidase and post synaptic acetylcholine receptor binding activity were carried out according to standard protocols. The results were analyzed using the standard ANOVA procedures. Results Among various solvent based leaf extracts of A. serpyllifolia tested, aqueous extract showed maximum neutralizing and inhibitory activities against Naja naja and Daboia russelli venoms. Conclusions The various in vitro enzymatic studies reveal that the aqueous leaf extract of A. serpyllifolia plant could inhibit most of the toxic enzymes of the Naja naja and Daboia russelli venoms which could be further confirmed by in vivo studies.

Currently, photobiomodulation therapy (PBMT) is gaining space in the scientific and clinical environment. To help elucidate the importance of irradiance, this study evaluated the effect of two different PBMT irradiances (3.5 and 90 mW/cm

Feae&rsquo, s viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

A 30-year-old man presented with altered sensorium following Russell’s viper bite, which was found to be secondary to intracranial hemorrhage secondary to venom-induced consumptive coagulopathy. He was managed conservatively with blood component transfusion and antivenom injection, and successfully discharged.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers, however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity, SVSPs, which deplete fibrinogen, and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection, however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

The 'Big Four' venomous snakes -A comprehensive analysis of the venom proteome composition of the 'Big Four' and other medically important venomous snakes of India and the effect of regional variation in venom composition on immunorecognition and/or neutralization by commercial antivenom was undertaken by searching the literature (from 1985 to date) available in large public databases. Further, mass spectrometric identification of poorly immunogenic toxins of snake venom (against which commercial polyvalent antivenom contains a significantly lower proportion of antibodies) and its impact on antivenom therapy against snakebite are discussed. The application of mass spectrometry to identify protein (toxin) complexes as well as drug prototypes from Indian snake venoms and the clinical importance of such studies are also highlighted.Further detailed clinical and proteomic research is warranted to better understand the effects of regional snake venom composition on the clinical manifestation of envenomation and antivenom therapy and to improve the production of antibodies against poorly immunogenic venom components.

Echis pyramidum (Epy) is a venomous snake belongs to Viperidae family; it causes fetal coagulopathy systemic effects and death. Searching for more effective and safe antivenom is mandatory for viper bites treatment. Proteases are the most lethal components in viper venom inducing hemorrhage, edema and coagulation problems. Thus, the study aims to evaluate the potency of the prepared antisera and their neutralizing properties against the biological activities induced by whole Epy venom individually. Echis pyramidum metalloprotease enzyme (60 kDa) was purified using size-exclusion followed by DEAE-ion exchange chromatography. The purified Epy metalloprotease enzyme (SVMP) was detoxified with 1.5 kGy gamma rays from cobalt60 gamma cell and used for immunization. 1.5 kGy irradiated Epy metalloprotease (SVMPi) showed less lethal activity (LD50) compared to the corresponding native immunogen. The prepared antisera boosted against whole Epy venom (WV), 1.5 kGy irradiated whole Epy venom (WVi), SVMP and SVMPi were tested for neutralization of lethality and biological activities induced by Epy venom. The antibodies elicited against WVi and SVMPi were 30,000 and 20,000 EU, respectively. The anti-SVMPi serum showed the highest neutralization of lethality (ED50) compared to the other prepared antisera. In addition, it prolonged the clotting time from 49.0 ± 2.5 to 176.2 ± 1.4 s. Furthermore, it demonstrated a highly neutralizing activity against edema induction and hemorrhage of Epy venom by 66.8% and 94.3%, respectively compared with the other prepared antisera. These findings would encourage further studies for using gamma irradiated purified fraction(s) from different snake venoms as safe antigen(s) to produce more effective antivenoms.

Macrovipera lebetina obtusa (MLO) is a venomous snake endemic to Middle East. Here we describe the therapeutic potential of the MLO snake venom. In S-180 sarcoma-bearing mouse model, we showed that the MLO snake venom inhibits tumour growth by 50%. In human dermal microvascular endothelial cells (HMVEC-D), treatment with the MLO snake venom lead to an increase of expression levels of the vascular endothelial growth factor (VEGF), while the level of the expression of caspase 8 did not change. In HMVEC-D cells MLO snake venom induces necroptosis, rather than apoptosis. In the chick embryo chorioallantoic membrane (CAM) assay, exposure to MLO snake venom inhibited bFGF-induced angiogenesis by 22%. Taken together, these results indicate that the MLO snake venom has a potent cytotoxic activity. Regulated necroptic cell death pathway, which is engaged by MLO snake venom, may become a promising novel target for antitumor therapies.

The saw-scaled viper (

Although envenoming by a small East European species of viper is rarely severe, and only exceptionally fatal, lack of specific antivenom stocks in a few areas within this region and possible severe side effects of antivenom application leave most bites to be treated only with antihistamines and supportive therapy. Varespladib is an effective inhibitor of snake phospholipase, and, as such, it could be considered as first-line therapy. The Nikolsky&rsquo, s viper venom contains an extremely high concentration of phospholipase A2 (PLA2), responsible for the toxic effects of the venom, as well as minor amounts of other toxins. If Varespladib can successfully inhibit PLA2 activity, the Nikolsky&rsquo, s viper could be one of the first venomous snakes having an antitoxin-specific treatment regimen. To assess that, Varespladib was administered alone subcutaneously to adult male CD-1 mice (8 mg/kg) and compared to mice exposed to Vipera berus nikolskii crude venom (8 mg/kg = 10 LD50) or a combination of Varespladib and the same amount of the venom. Experimental animals were monitored for the presence of envenoming symptoms and mortality for 48 h after injection. Eighty percent of mice receiving both Varespladib and venom survived, while 100% of the control group receiving venom alone died within 4 h. Experimental results are consistent with Varespladib acting as an effective antitoxin in the mouse model against Nikolsky&rsquo, s viper venom. Further studies are needed under experimental conditions that more closely resemble natural envenoming (i.e., delayed administration).

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell&rsquo, s viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

Snake bites are very common in India especially in rural areas. Empty sella syndrome is a relatively uncommon sequela of Russell's viper bite. Here, we describe a case of 22-year-old female who was seriously envenomed by Russell's viper bite at the age of 14 years. Diagnosis of hypopituitarism following Russell's viper bite was significantly delayed. We attribute her empty sella syndrome and hypopituitarism to Russell's viper envenoming.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and fiscal snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using multiple in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings strongly support the translation of combinations of safe and affordable enzyme inhibitors as novel broad-spectrum therapeutics for snakebite.

Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis. The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians’ morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis.

Background The pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1. Methods Male and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10−12 to 10−8 M) and sarafotoxin 6c (S6c, ETB agonist, 10−12 to 10−8 M) using the blood-perfused juxtamedullary nephron preparation. Results Control afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 μm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10−8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10−8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10−8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10−12 to 10−9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation. Conclusion These data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

Background: Envenoming by vipers Russell's and Echis Carinatus are common accidents faced by farmers and labors. Both viper venom toxins alter coagulation mechanism in the victim. The dose of snake antivenin to neutralize the venom is empirical and varies. Though the clinical manifestations in both vipers bite envenoming are nearly similar but dose of antivenin required is more in Russell's viper. We studied in detail about the correlation of clinical manifestations and confirmed species of snake. Methods: Cases of vipers snake bites admitted for last two successive years were studied. Analysis by local manifestations, systemic involvement, 20 minute whole blood clotting test (20WBCT), identification of snake responsible for clinical effects are confirmed by the snake species brought by victims or bystanders, in case of where victim saw the snake bur failed to kill. The victims identified from pictures of big four poisonous snakes (Russell's viper, Echis carinatus, Cobra, and krait). Further confirmation from the species responsible is done by showing the hospital preserved specimen to identify the culprits. Findings: About 77 cases of viper bite studied of these 57 has clinical syndrome suggestive of Russell's viper (RV) bite, one has dry bite, 23 victims brought the killed specimen confirmed RV, of these 18 identified the specimen picture and 5 wrongly locate the species in pictures, 20 victims correctly identified the hospital preserved specimen while 3 failed to recognize. There were 28 patients who saw the snake while bitten but failed to kill, of these 20 patients identify correctly the species on picture while 8 failed to identify. Only 22 correctly identified the culprit by looking at the hospital preserved specimen and 6 were confused. One dry bite victim correctly identified the bitten snake species in picture and hospital preserved specimen. Interpretation: In viper bite poisoning clinical pictures and hospital specimen help to confirm the species are highly supportive for clinical diagnosis.

Introduction: Russell’s viper envenoming in dogs is a significant problem in Sri Lanka. The current study focused on investigating clinical profile, laboratory findings of three selected tests and to develop a treatment strategy with Indian polyvalent Anti-Venom Serum (AVS). It was also intended to report adverse effects and complications caused by both Russell's viper venom (RVV) and AVS in Russell’s Viper (RV) envenomed dogs. Objective: To evaluate and report the clinical manifestations, to find out the minimum effective vials of AVS and to record AVS induced adverse reactions of RV envenoming in dogs. Materials and methods: A prospective study was conducted on Russell's viper bitten dogs (n = 65) admitted to the Veterinary Teaching Hospital (VTH) in Sri Lanka. Indian polyvalent AVS was used to treat all the envenomed dogs. The number of vials of AVS that was administered to a patient was decided upon by a second degree polynomial model with a number of vials of AVS in the X axis verses Prothrombine Time (PT), Activated Partial Thromboplastine Time (aPTT) and Clotting Time (CT) in the Y axis respectively. Results: Varying degrees of pain were exhibited by all the victim dogs. Mild swelling and necrosis at the site of bite was seen in 54% (n = 35) and 37% (n = 24) of dogs respectively. Prolonged values of, PT, aPTT and CT were seen from all the RV envenomed dogs. The mean leukocyte count in these dogs was 39.79 × 103/μL (normal range; 4–20 × 103/μL) (IQR:29.05 × 103/μL-45.92 × 103/μL). Statistical analysis showed that the initial vials of 7 AVS would be the minimum required vials. Therefore, a range of 6–15 AVS vials in total were administered to these dogs and in 7.6% (n = 5) of dogs, the results of PT, aPTT and CT became normal with 6 AVS vials at 32–97 minutes. Acute Renal Failure (ARF) was detected from 29% (n = 19) of dogs as a complication. Conclusions: Systemic clinical signs of haemorrhagic lesions, cardio respiratory toxicities were common in Russell's viper envenomed dogs. Initially 6 vials of AVS must be administered. AVS induced reactions were reported commonly. Russell's viper envenoming was found to be lethal in dogs. Keywords: Daboia russelii, Dogs, Anti-Venom serum, Prothrombine time, Activated partial thromboplastine time, Clotting time

Malayan pit vipers (Calloselasma rhodostoma) are major health hazard in Southern Thailand causing systemic bleeding by defibrination and thrombocytopenia. Twenty minute whole blood clotting test (20WBCT) is a useful and informative bedside test recommended by WHO for diagnosis of significant coagulopathy following snakebite envenoming since it to some extent predicts the need for anti-venom therapy. Prothrombin time (PT), international normalized ratio (INR) and activated partial thromboplastin time (APTT) are standard clotting assays that are widely used for diagnosis of coagulopathy. The purpose of this study is to compare 20WBCT with the standard clotting assays (PT, INR, APTT, fibrinogen) in management of Malayan pit viper bitten-patients. We studied 296 victims prospectively during a 3-year period. In total, 115 (38.9%) cases showed a positive 20WBCT (un-clot) suggesting systemic envenomation. Thrombocytopenia (platelet count less than 100,000/μL), prolonged PT, prolonged APTT, INR ≥ 1.2, and fibrinogen level100 mg/dl were found in 12.2%, 47.6%, 18.9%, 34.8% and 40.9%, respectively. Significant predictors for thrombocytopenia by multivariate analysis included systemic bleeding (p 0.001), higher INR (p 0.001), 20WBCT showing unclotted blood (p = 0.049), and longer bite-to-hospital time (p = 0.031). When a fibrinogen level below 100 mg/dl was determined as hypofibrinogenemic condition, INR had the highest area under ROC curve followed by 20WBCT and APTT, respectively, to identify such as hypofibrinogenemic condition. The diagnostic accuracy of 20WBCT and INR at or more than 1.155 were better than APTT at any cut-off points. INR1.155 had a sensitivity of 78.5% and specificity of 90.3% while 20WBCT had sensitivity of 81.0% and specificity of 90.3%. CONCLUSIONS: Both 20WBCT and INR are useful to assess coagulation abnormality in Malayan pit viper-bitten patients.

Patient: Male, 50-year-old Final Diagnosis: Snake bite induce thromotic microangiopathy Symptoms: Coagulopathy • hemolysis • renal failure • snake bite • thrombocytopenia Medication: — Clinical Procedure: Plasma exchange Specialty: Hematology Objective: Unusual clinical course Background: Consumption coagulopathy post envenomation is one the most common complications after a snakebite. It occurs secondary to activation of a coagulation cascade by snake venom and could be followed by a syndrome consistent with thrombotic microangiopathy. The efficacy of plasma exchange for the treatment of thrombotic microangiopathy post envenomation is a matter of debate. Case report: We reported the case of a 50-year-old male who had Arabian saw-scaled viper envenomation. He developed venom induced coagulopathy that improved within 24 hours of antivenom therapy. He subsequently developed micro-angiopathic hemolytic anemia, thrombocytopenia, and renal failure that was consistent with thrombotic microangiopathy. The patient was treated by plasma exchange and hemodialysis. He made a full recovery and was discharged after 4 weeks. Conclusions: This case report supports plasmapheresis as an option for management of a patient who develops thrombotic microangiopathy secondary to snake bite, especially those who do not improve with antivenom and supportive therapy.

The asp viper Vipera aspis aspis is a venomous snake found in France, and despite its medical importance, the complete toxin repertoire produced is unknown. Here, we used a venomics approach to decipher the composition of its venom. Transcriptomic analysis revealed 80 venom-annotated sequences grouped into 16 gene families. Among the most represented toxins were snake venom metalloproteases (23%), phospholipases A2 (15%), serine proteases (13%), snake venom metalloprotease inhibitors (13%) and C-type lectins (12%). LC-MS of venoms revealed similar profiles regardless of the method of extraction (milking vs defensive bite). Proteomic analysis validated 57 venom-annotated transcriptomic sequences (>70%), including one for each of the 16 families, but also identified 7 sequences not initially annotated as venom proteins, including a serine protease, a disintegrin, a glutaminyl-peptide cyclotransferase, a proactivator polypeptide-like and 3 aminopeptidases. Interestingly, phospholipases A2 were the dominant proteins in the venom, among which included an ammodytoxin B-like sequence, which may explain the reported neurotoxicity following some asp viper envenomations. In total, 87 sequences were retrieved from the Vipera aspis aspis transcriptome and proteome, constituting a valuable resource that will help in understanding the toxinological basis of clinical signs of envenoming and for the mining of useful pharmacological compounds. Biological significance The asp viper (Vipera aspis aspis) causes several hundred envenomations annually in France, including unusual cases with neurological signs, resulting in one death per year on average. Here, we performed a proteotranscriptomic analysis of V. a. aspis venom in order to provide a better understanding of its venom composition. We found that, as in other Vipera species, phospholipase A2 dominates in the venom, and the presence of a sequence related to ammodytoxin B may explain the reported neurotoxicity following some asp viper envenomations. Thus, this study will help in informing the toxinological basis of clinical signs of envenoming.

Pit viper envenoming is common in the hilly and the Himalayan regions of Nepal. Antivenom present in Nepal is unlikely to neutralize the venom of these pit vipers, although it has been used often by the healthcare providers in the clinical practice. Here, we report 15 cases of snakebite with a deranged coagulation profile. All patients recovered from envenoming on conservative management and without the administration of antivenom.

Lupus anticoagulants (LA) are heterogeneous antibodies and no single assay will detect every LA. Consequently, testing is commonly undertaken with both dilute Russell's viper venom time (dRVVT) and LA-responsive activated partial thromboplastin time (aPTT) to maximize detection rates. Although a huge body of evidence attests to the diagnostic utility of these assays, they have limitations that can render them unreliable in certain circumstances. Other assays are available for detecting LA but unfamiliarity, variable availability and technical concerns expressed in guidelines contribute to less usage than dRVVT and aPTT. However, assays such as Taipan snake venom time and Textarin time are insensitive to anticoagulants that compromise dRVVT and aPTT, and assays such as dilute prothrombin time can detect LA unreactive in dRVVT and aPTT. The pros and cons of alternative assays to dRVVT and APTT for LA detection are discussed.

Echis carinatus (EC) envenomation causes severe immune response by the accumulation of tissue debris in the form of DAMPs resulting in chronic inflammation and progressive tissue necrosis at the bitten site. Clearing of tissue debris is a prerequisite to enhance the healing of venom-induced necrotic wounds. Tricosanthus tricuspidata is a medicinal plant used extensively for the treatment of snake bite-induced toxicities. The active component responsible for the observed pharmacological action is a serine protease, tricuspidin. The topical application of tricuspidin was able to neutralize ECV-induced mouse footpad tissue necrosis and open wound in rabbits. Tricuspidin exerted its healing action via proteolytic activity as a consequence of upregulation of MMP-8 and down regulation of MMP-9. Further, tricuspidin reduced ECV-induced inflammation by decreasing the expression of TNF-α, IL-6 and MPO, and by increasing the level of VEGF-A and TGF-β1. The modulation of ECV induced immune/inflammatory mediators by tricuspidin was found to be more effective than trypsin. Moreover, tricuspidin and trypsin activated MAPKs via protease activated receptors-2 (PAR-2). These data indicate that the proteolytic activity of tricuspidin directly involved in the healing of ECV-induced chronic wound.

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell’s viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p

Herein, we report on the venom proteome of

The karst viper (Vipera ursinii ssp.) favours high-mountain dry grasslands in southern and south-eastern Croatia. It is medically less important than other Vipera species, because of its remote habitat and the very small amount of venom that it injects by its relatively short fangs. The scientific literature on Vipera ursinii deals mostly with the morphology, ecology and distribution range of this snake, due to the species’ conservation issues, while the toxinological aspects of its venom have not so far been investigated. Here we report on the composition and biological activity of the Vipera ursinii ssp. venom. Using a proteomics approach, we have identified 25 proteins in the venom that belong to seven protein families: snake venom metalloproteinase, serine protease, secreted phospholipase A2, cysteine-rich secretory protein, snake C-type lectin-like protein, serine protease inhibitor and nerve growth factor. The Vipera ursinii ssp. venom was found to be distinctively insecticidal. Its lethal toxicity towards crickets was more than five times greater than that of Vipera ammodytes ammodytes venom, while the opposite held in mice. Interestingly, the mode of dying after injecting a mouse with Vipera ursinii ssp. venom may suggest the presence of a neurotoxic component. Neurotoxic effects of European vipers have so far been ascribed exclusively to ammodytoxins and ammodytoxin-like basic secreted phospholipases A2. Structural and immunological analyses of the Vipera ursinii ssp. venom, however, confirmed that ammodytoxin-like proteins are not present in this venom.

Snake venoms contain components selected to immobilize prey. The venoms from Elapidae mainly contain neurotoxins, which are critical for rapid prey paralysis, while the venoms from Viperidae and Colubridae may contain fewer neurotoxins but are likely to induce circulatory disorders. Here, we show that the venoms from Protobothrops mucrosquamatus and Trimeresurus stejnegeri are comparable to those of Naja atra in prey immobilization. Further studies indicate that snake C-type lectin-like proteins (snaclecs), which are one of the main nonenzymatic components in viper venoms, are responsible for rapid prey immobilization. Snaclecs (mucetin and stejnulxin) from the venoms of P. mucrosquamatus and T. stejnegeri induce the aggregation of both mammalian platelets and avian thrombocytes, leading to acute cerebral ischemia, and reduced animal locomotor activity and exploration in the open field test. Viper venoms in the absence of snaclecs fail to aggregate platelets and thrombocytes, and thus show an attenuated ability to cause cerebral ischemia and immobilization of their prey. This work provides novel insights into the prey immobilization mechanism of Viperidae snakes and the understanding of viper envenomation-induced cerebral infarction.