Types of snake venom how many types

In addition, the venoms of viper snakes contain isoforms of group II PLA2s that are catalytically-active e. Figure 1. Structure of PLA2s from snake venoms. Green: N-terminal region critical for enzymatic and neurotoxic properties, C-terminal region essential for enzymatic activity and central Histidine in the catalytic site Rouault et al. D,E Highlighted in blue are the amino acids positions involved in the enzymatic, toxic and pharmacological properties of Crotoxin B Soares et al.

The central histidine in the catalytic site of Crotoxin B is highlighted in red. A number of PLA2s exert strong myotoxic effects which often lead to severe necrosis Harris and Maltin, ; Gutierrez and Ownby, , and many of these toxins also promote inflammation, including edema formation, cytokine production and leukocyte recruitment, pain by inducing thermal allodynia and mechanical hyperalgesia, paralysis through block of neuromuscular transmission and intensify hemorrhage by inhibiting coagulation Table 1 Camara et al.

Neurotoxic effects caused by these toxins, as well as some of their proinflammatory effects, occurs via the modulation of pre-synaptic terminals as well as sensory nerve-endings Camara et al. Overall, these pre-synaptic effects induce robust exocytosis of the neurotransmitters vesicles reserves which consequently lead to the depletion of neurotransmitter release in the neuromuscular junction to promote muscle paralysis Harris et al.

Table 1. Snake toxins and their multifunctional roles in the toxicity induced by snakebites. The inflammation induced by PLA2s has non-neurogenic and neurogenic substance-P dependent components Camara et al. The non-neurogenic component is mostly caused by the hydrolysis of membrane lipids that generate potent pro-inflammatory lipid mediators Costa et al.

Additional non-neurogenic and neurogenic inflammations induced by PLA2s use more complex mechanisms still not fully understood. For example, leukocyte recruitment De Castro et al.

Furthermore, substance-P mediated neurogenic inflammation has been described to be induced by PLA2s from Crotalus durissus cascavella Camara et al.

Interestingly, the C-terminal of Myotoxin-II a LysPLA2 isolated from Bothrops asper was able to activate macrophages, showing this region maybe be crucial for the observed enzymatic-independent inflammation Giannotti et al. The pain induced by PLA2s is driven by inflammatory processes and sensory neuronal activation. Bradykinin is an important mediator of the inflammatory pain induced by PLA2s Moreira et al. This suggests that PLA2s contribute to an increase in arachidonic acid release from cell membranes and its availability to be processed by cyclooxygenase resulting in prostaglandin production Verri et al.

Direct activation of sensory neurons was demonstrated by MitTx from Micrurus tener tener , a heteromeric complex between a PLA2 and a kunitz peptide Bohlen et al. This agonistic effect induces robust pain behavior in mice via activation of ASIC1 channels on capsaicin-sensitive nerve fibers Bohlen et al.

BomoTx also activated a cohort of sensory neurons to induce ATP release followed by activation of purinergic receptors Zhang et al. Unfortunately, the primary target of this neuronal activation is still unknown. The multifunctionality of PLA2s is evidenced by their myotoxic, neurotoxic and enzymatic functions, as well as by their inflammatory properties. There is evidence that separate domains and regions of the PLA2s structure participate in these various activities Figures 1A,B.

For example, for the LysPLA2 from Bothrops asper and Agkistrodon piscivorus piscivorus , the C-terminal region of these toxins residues — were identified as the active sites responsible for their myotoxic effects Lomonte et al. Interestingly, the same C-terminal region in BpirPLA2-I isolated from Bothrops pirajai had anticoagulant activity through inhibition of platelet aggregation Teixeira et al. Crotoxin B, an AspPLA2, and a major component of the venom of Crotalus durissus terrificus , has toxic active sites fully independent of its enzymatic activity Soares et al.

A detailed mutational study using the PLA2 OS2 from the Australian Taipan snake Oxyuranus scutellatus scutellatus revealed that a fold loss in enzymatic activity had only a minor effect on its neurotoxicity Rouault et al. Furthermore, the enzymatic activity of OS2 was dependent of the N- and C-terminal regions, and the N-terminal region had a major role in the central nervous system neurotoxicity.

In this study, the mutant ArgAla lost both nociceptive and edematogenic properties, LysAla and LysAla lost the nociceptive effects without interfering with the edema formation and LysAla lost the nociceptive properties and had weak inflammatory effects Figure 1E. Similarly, an independent study showed the LysAla substitution led to reduced membrane damaging and myotoxic activities Ward et al. This C-terminal region is characterized by the presence of basic and hydrophobic residues which have been strongly associated with the ability of PLA2s to interact and penetrate the lipid bilayer Delatorre et al.

Many snake venom toxins are known to be encoded by multi-locus gene families Casewell et al. The process of gene duplication and loss underpins the evolution of many snake venom toxin families, including the PLA 2 s Lynch, ; Vonk et al.

Indeed, studies have demonstrated that extremely divergent venom phenotypes e. It remains unclear as to the specific processes that underpin such diversity, although natural selection driven by environmental factors and hybridization events have both been proposed Dowell et al. These toxins are major components of viper venoms and play a key role in the toxicity of these snake venoms Table 1 ; Tasoulis and Isbister, The final class, P-I SVMPs which consist only of the metalloproteinase domain, appeared to have evolved on multiple independent occasions in specific lineages as a result of loss of the P-II disintegrin-encoding domain Casewell et al.

Throughout this diverse evolutionary history, SVMPs show evidence of extensive gene duplication events, coupled with bursts of accelerated molecular evolution Casewell et al. These abundance differences likely underpin the distinct pathologies observed following envenomings by snakes found in these families. SVMPs contribute extensively to the hemorrhagic and coagulopathic venom activities following bites by viperid snakes, and the diversity of SVMPs isoforms often present in their venom likely facilitate synergistic effects, such as simultaneous action on multiple steps of the blood clotting cascade Kini and Koh, ; Slagboom et al.

However, it is relatively uncommon for elapid snakebites to cause systemic hemotoxicity Slagboom et al. Figure 2. Structure of metalloproteinaises from snake venoms. Cysteines are colored in red, the disintegrin-like domain is highlighted in green and the cysteine-rich domain is highlighted in blue.

The metalloproteinase domain is colored in orange, the disintegrin-like domain D-like is colored in green and the cysteine-rich domain Cys-rich is colored in blue. The disulphide bridges are colored in yellow. Research has revealed that the effects of SVMP-induced hemorrhage relies on a mechanism that occurs in two steps Gutierrez et al. First, SVMPs cleave the basement membrane and adhesion proteins of endothelial cells-matrix complex to weaken the capillary vessels.

During the second stage, the endothelial cells detach from the basement membrane and become extremely thin, resulting in disruption of the capillary walls and effusion of blood from the fragile capillary walls.

In addition to the proteinase activity, SVMPs impact on homeostasis by altering coagulation, which contributes to their toxic hemorrhagic effects Markland, ; Takeda et al. This occurs through modulation of factors such as fibrinogenase and fibrolase that mediate the coagulation cascade, depletion of pro-coagulation factors through consumption processes e.

Some SVMPs also induce inflammation, including edema, and pain by triggering hyperalgesia Dale et al. Neurogenic inflammation was also implicated in the local hemorrhage induced by Bothrops jararaca which was shown to be dependent on serotonin and other neuronal factors Goncalves and Mariano, The mechanisms on how neurogenic inflammation is triggered by the snake venom components and how it participates in the hemorrhagic process are still not understood.

Pain induced by SVMPs is characterized by hyperalgesia and inflammatory pain, which is dependent on the production of cytokines, nitric oxide, prostaglandins, histamine, leukotrienes, and migration of leukocytes, mast cell degranulation and NFkB activation Fernandes et al. However, the mechanisms underlying SVMP-induced pain are still poorly understood, with neurogenic inflammation and neuronal excitatory properties still underexplored. The multifunctional properties of SMVPs are also well-described.

These observations suggest that these domains are involved in the inflammatory hyperalgesia induced by SVMPs. Furthermore, the pronounced hemorrhagic and necrotic activities are strongly dependent on biological effects driven by the disintegrin-like and cysteine-rich domains, as observed for BJ-PI2 da Silva et al. The hemorrhagic activity of Bothrops jararaca venom was also shown dependent on neurogenic inflammation Goncalves and Mariano, These venom toxins have evolved from kallikrein-like serine proteases and, following their recruitment for use in the venom gland, have undergone gene duplication events giving rise to multiple isoforms Fry et al.

SVSPs catalyze the cleavage of polypeptide chains on the C-terminal side of positively charged or hydrophobic amino acid residues Page and Di Cera, ; Serrano, Whilst the SVMPs are well-known for their ability to rupture capillary vessels, SVSPs execute their primary toxicity by altering the hemostatic system of their victims, and by inducing edema and hyperalgesia through mechanisms still poorly understood Table 1.

Hemotoxic effects caused by SVSPs include perturbations of blood coagulation pro-coagulant or anti-coagulant , fibrinolysis, platelet aggregation and blood pressure, with potential deadly consequences for snakebite victims Murakami and Arni, ; Kang et al.

Figure 3. Structure of Serine proteinases from snake venoms. For example, the activation of prothrombin produces thrombin which in turn produces fibrin polymers that are cross-linked. Thrombin also activates aggregation of platelets which, together with the formation of fibrin clots, results in coagulation Murakami and Arni, In addition, platelet-aggregating SVSPs will activate the platelet-receptors to promote binding to fibrinogen and clot formation Yip et al.

These procoagulant and platelet-aggregating activities will lead to the rapid consumption of key factors in the coagulation cascade and clot formation. Furthermore, fibrinolytic SVSPs play an important role in the elimination of blood clots by acting as thrombin-like enzymes or plasminogen activators, which eliminates the fibrin in the clots and contributes significantly to the establishment of the coagulopathy Kang et al. Little is known about inflammatory responses and hyperalgesia induced by SVSPs.

SVSPs in the venoms of Bothrops jararaca and Bothrops pirajai induce inflammation through edema formation, leucocyte migration mainly neutrophils and mild mechanical hyperalgesia, however, the mediators involved in these effects are still unknown Zychar et al. Three-fingers toxins 3FTXs are non-enzymatic neurotoxins ranging from 58 to 81 residues that contain a three-finger fold structure stabilized by disulfide bridges Osipov and Utki, ; Kessler et al.

They are present mostly in the venoms of elapid and colubrid snakes, and exert their neurotoxic effects by binding postsynaptically at the neuromuscular junctions to induce flaccid paralysis in snakebite victims Barber et al. Furthermore, they can exist as monomers and as covalent or non-covalent homo or heterodimers. The diversity of 3FTX isoforms described above are a direct result of a diverse evolutionary history, whereby ancestral 3FTXs have diversified by frequent gene duplication and accelerated rates of molecular evolution.

These processes, which are broadly similar to those underpinning the evolution of the other toxin families described above, are particularly associated with the evolution of a high-pressure hollow-fanged venom delivery system observed in elapid snakes Sunagar et al.

For example, gene duplication events have resulted in the expansion of 3FTX loci from one in non-venomous snakes like pythons, to 19 in the elapid Ophiophagus hannah king cobra Vonk et al. The consequences of this evolutionary history are the differential production of numerous 3FTX isoforms that often exhibit considerable structural differences and distinct biological functions Figures 4B—E.

Although many elapid snakes exhibit broad diversity of these functionally varied toxins in their venom e. Figure 4. Structure of three-finger toxins from snake venoms.

K Neurotoxin II from N. L Neurotoxin b NTb from O. Despite the shared three-finger fold, the 3FTXs have diverse targets and biological activities. Their toxic biological effects include flaccid or spastic paralysis due to the inhibition of AChE and ACh receptors Grant and Chiappinelli, ; Changeux, ; Marchot et al. In addition to their multitude of bio-activities, 3FTXs can remarkably display toxicities that target distinct classes of organisms as demonstrated in non-front fanged snake venoms that produce 3FTX isoforms which are non-toxic to mice but highly toxic to lizards, and vice-versa Modahl et al.

Furthermore, 3FTXs are relatively small compared to the other snake toxins discussed herein, and do not exhibit multiple domains to produce their multiple toxic functions. Nevertheless, the number of receptors, ion channels, and enzymes targeted by snake 3FTXs highlights the unique capacity of this fold to modulate diverse biological functions and the arsenal of toxic effects that are induced by 3FTXs.

The unique multifunctionality of the 3FTX scafold occurs because of their resistance to degradation and tolerance to mutations and large deletions Kini and Doley, Therefore, the structure-activity relationship of the 3FTXs is complex and yet to be fully understood. Their functional sites are located on various segments of the molecule surface. Conserved regions determine structural integrity and correct folding of 3FTXs to form the three loops, including eight conserved cysteine residues found in the core region.

Additional disulfide bonds can be observed either in the loop I or loop II which can potentially change the activity of the 3FTX in some cases. Specific amino acid residues in critical segments of the 3FTXs have been identified to be important for binding to their targets. For example, the interactions between fasciculin and AChE enzyme has been studied.

The first loop or finger of fasciculin reaches down the outer surface of the enzyme, while the second loop inserts into the active site and exhibit hydrogen bonds and hydrophobic interaction Harel et al. Several basic residues in fasciculin make key contacts with AChE.

From docking studies, hydrogen bonds, and hydrophobic interactions where shown to establish receptor-toxin assembly. Hydrophobic interactions are also observed between eight amino acid residues Lys32, Cys59, Val34, Leu48, Ser26, Gly36, Thr15, Asn20 from fasciculin and the enzyme active site Waqar and Batool, These interactions involve charged residues but lacks intermolecular salt linkages.

Muscarinic toxins from mamba venoms, such as MT1 and MT7 Figures 4G,H , act as highly potent and selective antagonists of M1 receptor subtype through allosteric interactions with the M1 receptor.

Fruchart-Gaillard et al. In this study, substitution within loop 1 and loop 3 weaken the toxin interactions with the M1 receptor, resulting in a 2-fold decrease in affinity Figures 4I,J.

Furthermore, modifications in loop 2 of the MT1 and MT7 significantly reduce the affinity for the M1 receptor. These two residues were not located at the tip of the toxin loop, however, they played a critical role in the interactions with their molecular targets Bourne et al. The insertion of the loop II into the binding pocket of a nAChR induces the neurotoxin activity and significantly determines the toxin-receptor interactions, while loop I and III contact the receptor residues by their tips only and determine the immunogenicity of the short neurotoxins.

The structure of neurotoxin b NTb , a long neurotoxin from Ophiophagus hannah , has been elucidated Peng et al. Conserved residues in loop II also play an important role in the toxicity of the long neurotoxins by making ionic interactions between toxin and receptor. Positively charged residues Trp27, Lys24 and Asp28 are highly conserved residues in the long neurotoxins. Furthermore, a modification of the Trp27 in the long neurotoxin analog of NTb from king cobra venom led to a significant loss in neurotoxicity.

The additional disulphide bridge in loop II of long neurotoxins does not affect the toxin activity. Nevertheless, cleavage of the additional disulphide bridge in loop II can disrupt the positively charged cluster at the tip of loop II. Changes in loop II conformation will affect the binding of the long neurotoxin to the target receptor resulting the loss of neurotoxicity Peng et al. Long and short neurotoxins show sequence homology and similar structure. Previous studies show that many residues located at the tip of loop II are conserved in both short and long neurotoxins.

However, significant differences between long-chain neurotoxin and short chain neurotoxin are indicated by the immunological reactivity. Many of the residues involved in the antibody-long neurotoxins binding are located in loop II, loop III, and in the C-terminal, while in short neurotoxins the antibody's epitope makes interactions with the loop I and loop II Engmark et al. Animal-derived antivenoms are considered the only specific therapy available for treating snakebite envenoming Maduwage and Isbister, ; Slagboom et al.

These consist of polyclonal immunoglobulins, such as intact IgGs or F ab' 2 , or Fab fragments Ouyang et al. Antivenoms can be classified as monovalent or polyvalent depending on the immunogen used during production.

Monovalent antivenoms are produced by immunizing animals with venom from a single snake species, whereas polyvalent antivenoms contain antibodies produced from a cocktail of venoms of several medically relevant snakes from a particular geographical region.

Polyvalent antivenoms are therefore designed to address the limited paraspecific cross-reactivity of monovalent antivenoms by stimulating the production of antibodies against diverse venom toxins found in different snake species, and to avoid issues relating to the wrong antivenom being given due to a lack of existing snakebite diagnostic tools O'leary and Isbister, ; Abubakar et al.

However, polyvalent therapies come with disadvantages—larger therapeutic dose are required to effect cure, potentially resulting in an increased risk of adverse reactions, and in turn increasing cost to impoverished snakebite victims Hoogenboom, ; O'leary and Isbister, ; Deshpande et al.

Variation in venom constituents therefore causes a great challenge for the development of broadly effective snakebite therapeutics. The diversity of toxins found in the venom of any one species represents considerable complexity, which is further enhanced when trying to neutralize the venom of multiple species, particularly given variations in the immunogenicity of the multi-functional toxins described in this review.

Antivenom efficacy is therefore, typically limited to those species whose venoms were used as immunogens and, in a number of cases, closely-related snake species that share sufficient toxin overlap for the generated antibodies to recognize and neutralize the key toxic components Casewell et al.

Because variation in venom composition is ubiquitous at every level of snake taxonomy e. Such studies have revealed surprising cross-reactivity of antivenoms against distinct, non-targeted, snake species, such as: i the potential utility of Asian antivenoms developed against terrestrial elapid snakes at neutralizing the venom toxicity of potent sea snake venoms Tan et al.

The later of these studies demonstrated cross-neutralization between distinct snake lineages e. Thus, detailed knowledge of venom composition can greatly inform studies assessing the geographical utility of antivenoms.

Such studies have stimulated much research into the development of novel therapeutic approaches to tackle snakebite. These include the use of monoclonal antibody technologies to target key pathogenic toxins found in certain snake species Laustsen et al. It is anticipated that in the future these new therapeutics may offer superior specificities, neutralizing capabilities, affordability and safety over conventional antivenoms. However, the translation of their early research promise into the mainstay of future snakebite treatments will ultimately rely on further research on the toxins that they are designed to neutralize.

Specifically, the selection, testing and optimization of new tools to combat snake envenoming is reliant upon the characterization of key pathogenic, and often multifunctional, toxins found in the venom of a diverse array of medically important snake species. The first drug derived from animal venoms approved by the FDA is captopril, a potent inhibitor of the angiotensin converting enzyme sACE used to treat hypertension and congestive heart failure Cushman et al.

Captopril was derived from proline-rich oligopeptides from the venom of the Brazilian snake Bothrops jararaca Ferreira et al. This milestone in translational science in the late 70's revealed the exceptional potential of snake venoms, and possibly other animal venoms such as from spider and cone snails, as an exquisite source of bioactive molecules with applications in drug development. More recently, an anti-platelet drug derived from the venom of the southeastern pygmy rattlesnake Sistrurus miliarius barbouri was commercialized as Integrillin by Millenium Pharmaceuticals, and is used to prevent acute cardiac ischemia Lauer et al.

The resulting product is now commercialized as Syn-AKE. Snake toxins have been applied with great success in diagnostics.

Snake toxins also have the potential to become novel painkillers. These findings, alongside current research into venom toxins, suggest an exciting future for the use of snake venoms in the field of drug discovery. Snake venoms are amongst the most fascinating animal venoms regarding their complexity, evolution, and therapeutic applicability. They also offer one of the most challenging drugs targets due to the variable toxin compositions injected following snakebite.

The multifunctional approach adopted by the major components of their venoms, by using multidomain proteins and peptides with promiscuous folds e. Gaining a better understanding of the evolution, structure-activity relationships and pathological mechanisms of these toxins is essential to develop better snakebite therapies and novel drugs. Recent developments in genomics, proteomics and bioactivity assays, as well as in the understanding of human physiology in health and disease, are enhancing the quality and speed of research into snake venoms.

We hope to improve the therapies used to neutralize the toxic effects of PLA2s, SVMPs, SVSPs and 3FTXs, and to develop drugs as new antidotes for a broad-spectrum of snake venoms that could also be effective in preventing the described inflammatory reactions and pain induced by snakebite. Finally, a diversity of biological functions in snake venoms is yet to be explored, including their inflammatory properties and their intriguing interactions with sensory neurons and other compartments of the nervous system, which will certainly lead to the elucidation of new biological functions and the development of useful research tools, diagnostics and therapeutics.

FC provided theme, scope, and guidance. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abubakar, I. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper Echis ocellatus envenoming in Nigeria. PLoS Negl. A standard curve with different concentrations of hyaluronic acid was used to determine the optimum concentration of the enzyme, and acetate buffer was used as the blank.

The absorbance was read at nm using an Epoch 2 microplate spectrophotometer. Snake venom protease activity of the crude venom was assayed using a protocol described previously, with modifications [ 11 ]. Azocaesin SRL, India in 0.

The supernatant was carefully collected and equal volumes of 0. The relative activity of crude venoms to the purified protease from bovine pancreas Sigma-Aldrich, USA; positive control was plotted. All reactions were performed in triplicates.

The fibrinogenolytic activity of the venoms were visualised using fibrinogen from human plasma Sigma-Aldrich, USA and applying a modified version of a method previously described [ 12 ]. The assay mixture was subjected to 0. Ltd, India was used as the positive control. The protocol was standardized by modifying a method described earlier [ 13 ]. To understand the effect of venom on the blood coagulation pathway, prothrombin time PT and activated partial thromboplastin time aPTT were assayed, which correspond to the time taken for the appearance of the first fibrin clot via extrinsic and intrinsic coagulation pathways, respectively.

Blood donated by healthy male volunteers, collected in a tube with 3. Haemolytic activities of venoms were assayed in triplicates using the in vitro method described earlier with slight modifications [ 15 ].

These reaction mixtures were then centrifuged at 15, x g for 5 mins at 4 0 C, followed by the measurement of absorbance of the supernatant at nm using an Epoch 2 microplate spectrophotometer. To evaluate capabilities of the currently marketed Indian polyvalent antivenoms in recognising and binding to epitopes on venom proteins, we conducted endpoint titration enzyme-linked immunosorbent assay ELISA , following a protocol described earlier with slight modifications [ 16 ].

Venom samples ng in carbonate buffer pH 9. The wells were rinsed 6 times with Tris-buffered saline 0. The optical density was measured at a wavelength of nm. The assay was performed in triplicates and the mean values were used for calculations. Purified equine IgG Bio-Rad Laboratories, USA was used as the negative control, and absorbance values above the cut off, calculated as the mean absorbance of the negative control plus two times the standard deviation, were considered for comparisons.

To visualise venom-antivenom antigen-antibody interactions, we performed immunoblotting experiments. The membrane was then incubated overnight with the marketed antivenom, diluted to concentrations in the blocking solution, following which it was incubated with HRP-conjugated, rabbit anti-horse secondary antibody dilution at RT for 2 hours.

Following the addition of substrate solution containing 0. Toxicity profiles of the venoms were determined using male CD-1 mice 18—22 g as per previously described methods and WHO guidelines [ 17 ]. After 24 hours of observation, death and survival patterns were recorded, and the LD 50 value was calculated using Probit statistics [ 18 ].

Four different antivenom dilutions were challenged against venom concentration five times that of the LD 50 value determined above for each venom. Venoms were incubated with four dilutions of antivenom at 37 0 C for 30 minutes, followed by intravenous injection into tail vein of five CD-1 mice per dilution.

The median effective dose of antivenom was calculated using Probit analysis, after 24 hours of observation. For N. Following the completion of these experiments, antivenom potency was expressed in milligrams of venom neutralised per millilitre of antivenom using the following equation. Venoms of these snakes were found to contain a wide range of low- and high-molecular weight toxins, and exhibited profound compositional diversity both inter- and intra-specifically.

SDS-PAGE venom profiles of snakes from the genera Naja , Bungarus and Echis revealed several unique bands of diverse molecular weights and intensities, highlighting the primary differences in their venom composition and relative abundance, respectively Fig 1C. While subtle differences in band intensities were observed in the venoms of Naja spp. In order to get a better understanding of the observed proteomic diversity, the venoms were subjected to tandem mass spectrometric analyses.

These analyses resulted in the identification of 51—59 proteins in the crude venoms of each Naja spp. Neurotoxic 3FTxs belong to the 3FTx superfamily of non-enzymatic venom proteins that consist of short-length 60—75 residues toxins, capable of exerting a broad range of clinical effects [ 21 ]. Surprisingly, more than three quarters of the venom of N. Contrastingly, the Arunachal Pradesh population of N.

Venoms of N. Other major venom components detected in Naja spp. For example, when compared to the two populations of N.

Similarly, N. The other notable difference observed in the venom proteomes of these species was related to Kunitz peptides. Venom compositions of Naja A , Echis B and Bungarus C species are depicted as pie charts, and the relative composition of toxins are indicated in percentiles. A unique colour key for individual toxins is provided. Surprisingly, while venom composition remained fairly similar, notable differences in relative abundance of various toxins were observed between the two subspecies.

PLA 2 constituted a major proportion On the contrary, LAAO The proteomic analyses of crude venoms of the Bungarus spp. Similar to the cobra species analysed here, the venoms of Bungarus spp. While PLA 2 constituted nearly three quarters of B. The relative abundance of neurotoxic 3FTxs was found to be fairly similar across the Bungarus spp.

Acetylcholinesterases AChEs constituted the third major family of venom proteins in B. Compositional differences in snake venoms have been shown to have profound implications on the pathologies that present in snakebite victims [ 23 — 25 ]. PLA 2 is amongst the most important snake venom toxin superfamilies, and the venoms of many Elapidae and Viperidae snakes are enriched with this toxin type [ 26 ]. The abundance and type of PLA 2 toxins can substantially alter the clinical profile of the venom [ 27 ].

Our results revealed significant differences in PLA 2 catalytic activities Fig 3A ; S1 Fig , where, at the highest venom concentrations tested 0. Interestingly, at very low venom concentrations 0.

At the lowest venom concentration 0. However, on increasing the concentration 0. Echis venoms exhibited very little to no PLA 2 enzymatic activity at lower venom concentrations 0.

All assays, with the exception of E and F , were performed in triplicates and the standard deviation is indicated by error bars. A colour code is provided, which corresponds to the respective species of snake. While both N. In contrast, all Bungarus spp. While both B. Following envenomation, hyaluronidase activity is considered critical for the diffusion of toxins from the bite site into the circulatory system. While E. Proteases, such as snake venom serine protease and snake venom metalloproteases, are amongst the medically important toxins that are commonly identified in venoms of many snake species [ 32 ].

They exert toxicity by targeting plasma proteins involved in the blood coagulation cascade, fibrinolysis, platelet aggregation, haemorrhage, etc [ 32 ]. Therefore, in order to understand the proteolytic nature of venoms, we performed colorimetric protease assays using azocaesin as substrate. While the elapid snake venoms of Naja and Bungarus spp. Interestingly, the venom of E. Several components of snake venoms have been shown to affect haemostasis by acting on various factors involved in the blood coagulation pathway [ 33 ].

With the exception of venom from B. Snake venoms comprise of toxins that cause cell destruction and necrosis, which, in turn, results in the extrusion of DNA and the formation of extracellular traps [ 34 , 35 ]. In addition to blocking blood vessels, these traps can result in the localized accumulation of tissue destroying toxins [ 34 , 35 ].

However, DNase, an enzymatic snake venom toxin with endonuclease activity [ 36 , 37 ], has been shown to prevent the entrapment of these toxins in the extracellular traps [ 34 ]. Thus, the presence or absence of DNase might significantly alter clinical manifestations post envenoming.

On the contrary, the venom of N. Interestingly, while N. A similar trend was observed in Bungarus spp. Although ATPase and ADPase enzymatic toxins are omnipresent in most snake venoms as minor toxin components, their pharmacological roles are not clear yet. The release of purines, mainly adenosine, has been theorized to play a role in inducing an array of clinical manifestations [ 38 ].

In this study, Naja spp. In a complete contrast, all three Bungarus spp. Many snake species are known to have a clinically significant impact on the blood coagulation cascade post envenomation. The venom toxins act on various blood coagulation factors, thereby, affecting blood coagulability, ultimately facilitating other toxins to inflict symptoms such as damaged blood vessels, shock, intracranial and pituitary haemorrhage, renal failure, thrombosis, pulmonary embolism and thrombocytopenia [ 39 — 41 ].

Bearing the significance of these clinical manifestations in mind, we estimated PT and aPTT to understand the effect of venoms under study on the extrinsic and intrinsic blood coagulation pathways, respectively Fig 3E and 3F ; S3A—S3C Table. Similar, anticoagulatory effects were observed in the case of B.

In contrast, the venoms of Naja and Bungarus species showed only minor deviance from the control values in PT estimation, with INR values being slightly more than 1.

This suggests that these venoms do not target the extrinsic or the common pathway of the clotting cascade [ 42 ]. PLA 2 s are amongst snake venom toxins known to cause direct haemolysis of erythrocytes by catalysing the hydrolysis of membrane phospholipids [ 43 , 44 ]. These experiments revealed the maximal haemolytic effects of N. While the venom of B. The absorbance values at nm, which directly correlate to the binding efficiency of antivenom, were plotted against the dilution factors.

For instance, all four commercial antivenoms tested recognized N. In the case of E. Most surprisingly, all antivenoms were found to possess poor binding capabilities against the venoms of the three Bungarus spp. It should be noted that the estimation of total IgG content in a vial of commercial antivenom revealed that the overall IgG content remains roughly the same across all four manufacturers 6.

This indicates that the differences observed in absorbance for various antivenoms is suggestive of the actual differences in binding efficiencies, and do not result from the differences in IgG contents. Optical density at nm is plotted against various dilutions of antivenoms. The values are provided as mean absorbance of triplicates, and error bars represent standard deviation. The dotted lines represent antivenom titres, which were determined using purified IgG from unimmunized horses as negative control.

Further, by employing immunoblotting experiments, we were able to identify several major venom components that are not effectively recognized by the marketed antivenoms Fig 5A—5F. For example, with the exception of Premium Serums, one of the bands at 55 kDa was not recognized by any of the antivenoms. Comparison of these immunoblots to the negative control naive horse IgG reveals non-specific binding of IgGs to these largely abundant low molecular weight toxins, especially in Elapidae snakes Fig 5B.

Results of immunoblotting experiments largely corroborate findings of indirect ELISAs and highlight the poor venom recognition potential of commercial antivenoms. This figure depicts the results of western blotting experiments. Therefore, we conducted WHO-recommended preclinical neutralization assays in mice.

These experiments revealed several fascinating aspects about the nature of venom in these medically important snakes. In the mouse model, the venom of N. Interestingly, E. These experiments estimated an LD 50 of 0. A and B depict the median lethal doses of various medically important snakes and the neutralising potency of Premium Serums antivenom, respectively. Vertical solid and dotted lines in panel B indicate marketed neutralising potencies of antivenoms against N.

In order to significantly reduce the number of mice sacrificed in these experiments, we only tested Premium Serums antivenom, which is amongst the most widely marketed polyvalent antivenoms in India. These experiments highlighted the inefficiencies of this commercial Indian antivenom in neutralising bites from several neglected snake species S4B Table. Some sources claim proteolytic venom as a fourth category of venom, however because all venom has proteolytic effects, it is not entirely correct to place it in its own category.

Effects: Paralysis, convulsions or rapid muscle twitching, difficulty breathing and other respiratory issues Deadly? In many cases, yes. The word neurotoxic comes from its effects on the nervous system. Neurotoxic venom essentially acts as a poison to the nervous system. The nervous system depends on neurotransmitters chemical signals and neurotransmitter receptors points where neurotransmitters bind to to send signals between the brain and our bodies. When neurotoxic venom is introduced into the body, it quickly causes problems.

Neurotoxic venom can reduce the production of neurotransmitters or block neurotransmitters all together, severely disrupting processes in the nervous system. These disruptions can essentially paralyze the muscles that we use to breathe, which in turn can cause respiratory failure and prevent bite victims from breathing.

In some cases, neurotoxic venom can overstimulate neurotransmitters, which can lead to rapid muscle twitching or convulsions. Survivors of snake-bites with neurotoxic venom do not tend to have lasting symptoms other than scarring around the bite site. Effects: Severe pain, swelling of area surrounding bite, necrosis death of tissue Deadly? Yes, but less-so than the other types of venom.

Cytotoxic venom affects the cells that make up tissues, organs and muscles in our body. Cytotoxic venom works quickly to kill and damage the body cells. Victims that are bitten by snakes with cytotoxic venom begin to experience the effects almost immediately.

Cytotoxic bites kill the tissues of the body, causing necrosis. Cytotoxins help to digest and break down prey before it is eaten, and in snake bite victims, the tissues around the bite site may be affected similarly and become liquified. With time, necrosis can spread from the bite site to other parts of the body. Those that are not treated right away may need to have the affected part of the body amputated to stop the spread of necrosis.

Lasting effects of a bite from a cytotoxic venom often include permanent tissue damage. Hemotoxic venom poisons the circulatory system or bloodstream. Once hemotoxic venom enters the blood stream, it begins to attack and kill red blood cells.

The red blood cells burst open and essentially prevent the blood clotting coagulation that naturally occurs in the body. Severe internal bleeding occurs as a result of blood cells rupturing and then the inability for the blood to clot. When the damaged red blood cells begin to accumulate or build up, it can prevent the kidneys from functioning properly.

Additionally, hemotoxic venom can also cause blockages in blood vessels which can lead to heart failure. Effects of hemotoxic venom typically take longer to affect the victim than neurotoxic or cytotoxic venom. Like all venomous snake bites, getting to treatment as quick is possible gives the victim the best chance of survival. Most snakes with neurotoxic venom are in the elapidae family, making them elapids. Example of a neurotoxic snake: King Cobra. Workers are more likely to be bitten when they unknowingly step on or near a copperhead.

Giving antivenom to a snake bite patient as soon as possible helps limbs to recover faster and it lessens the chance that a limb will be disabled after copperhead snake envenomation.

Adult cottonmouth snakes average 50—55 inches long. Juveniles have a bold cross-banded pattern of brown or orange with a yellow tail.

Cottonmouths are often found in or around water. Geographic Region: Wetland areas, rivers, lakes, etc. Other venomous snakes display warning coloration. These snakes are sometimes confused with nonvenomous king snakes, which have similar colored bands, but arranged differently. Coral snakes tend to hide in leaf piles or burrow into the ground.

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