Investigating myotoxicity following Australian red-bellied black snake (Pseudechis porphyriacus) envenomation.

BACKGROUND: Myotoxicity is one of the common clinical manifestations of red-bellied black snake (Pseudechis porphyriacus) envenomation characterised by elevated creatine kinase (CK) concentrations of greater than 1000 U/L. This study aimed to investigate the occurrence of myotoxicity in patients following envenomation. METHODS/PRINCIPAL FINDINGS: Patient characteristics and serial blood samples (timed venom concentrations and CK concentrations, pre- and post- antivenom) from 114 patients (median age 41, 2-90y; 80 male) were extracted from the Australian Snakebite Project database. Patients were categorised into three groups based on peak CK concentrations [no myotoxicity (<1000 U/L), mild (1000-10,000 U/L) and severe (>10,000 U/L)]. The odds of (mild or severe) myotoxicity was lower in patients that received early antivenom (within 6 hours post-bite) compared to those that received late or no antivenom (odd ratio was 0.186; 95% confidence interval, 0.052-0.664). A population pharmacokinetic-pharmacodynamic (PKPD) model was developed to describe the relationship between the time course of venom (a mixture of toxins) and effect (elevated CK). In addition, a kinetic-pharmacodynamic (KPD) model was developed to describe the relationship between time course of a theoretical toxin and effect. Model development and parameter estimation was performed using NONMEM v7.3. No single set of parameter values from either the PKPD or KPD models were found that could accurately describe the time course of different levels of severity of myotoxicity. The predicted theoretical toxin half-life from the KPD model was 11 ± 3.9 hours compared to the half-life of venom of 5.3 ± 0.36 hours. This indicates that the putative causative toxin's concentration-time profile does not parallel that of venom. CONCLUSION: Early antivenom administration reduces the incidence of myotoxicity. The venom concentration profile does not appear to be the driver for myotoxicity following envenomation. Additional factors that affect the sensitivity of the patient to snake venom/toxins must be explored to understand the relationship with myotoxicity.

The clinical course and treatment of black mamba (Dendroaspis polylepis) envenomations: a narrative review.

CONTEXT: The black mamba (Dendroaspis polylepis) is, due to its extremely toxic venom, one of the most dangerous snake species in Sub-Saharan Africa. A D. polylepis bite is a medical emergency and requires adequate action to prevent severe complications. However, there are no comprehensive reviews available based on clinical cases, and no readily accessible guidelines for standardized treatment. Therefore, we aim to provide an overview regarding the currently available clinical literature on D. polylepis envenomations; in order to promote knowledge on symptomatology and treatment options. METHODS: We searched for cases reporting humans bitten by D. polylepis in PubMed, Embase, Scopus, and Sabinet. We searched the reference lists of all eligible articles for additional articles. After quality assessment, 29 cases were included in this review. We used descriptive analysis to create an overview of the collected parameters. DISCUSSION: Among the included case reports and case series, D. polylepis envenomations most frequently resulted in decreased respiratory function, sweating and paralysis. The onset of symptoms usually occurred within 60 minutes. Neurological symptoms occurred more often than symptoms of autonomic dysfunction. In the reported cases most patients (26/29) received antivenom and most survived (25/29). We recommend the reporting of additional structured case reports to improve future analyses on the clinical course of envenomations, in order to improve public health response to D. polylepis envenomations.

A Quest for a Universal Plasma-Derived Antivenom Against All Elapid Neurotoxic Snake Venoms.

This review describes the research aimed at the development of universal antivenom against elapid neurotoxic snake venoms. The antivenoms produced in Thailand in the 1980s were of low potency, especially against the elapid venoms. This was thought to be due to the low immunogenicity of the α-neurotoxins, which are the most lethal toxins in these venoms. Comparisons of various α-neurotoxin conjugates and polymers, and also different immunological adjuvants, showed that the adjuvant used is the major determinant in the antibody response in horses. The potent Freund's adjuvant was not used due to its severe local side-effect in horses. Therefore, a novel immunization protocol termed 'low dose, low volume multi-site' was developed for use in horses. This immunization protocol has led to the production of highly potent monospecific antivenoms against several elapid and viperid venoms, and two potent polyspecific antivenoms, one against 4 neurotoxic and another against 3 hematotoxic venoms. The immunization protocol has also led to other improvements in antivenom production including: several fold increases in antiserum potency, a reduction in the time required to reach therapeutically useful antibody titers, a 90% reduction in the amount of venom used, and 100% of the horses responding to the immunization program. This development is partly responsible for significant decrease in the Thailand's annual snakebite death toll from a few dozens to mostly nil in recent years. Finally, a simple and novel immunization strategy, using a 'diverse toxin repertoire' composed of numerous elapid toxin fractions as immunogen, was proposed and tested. This immunization procedure has resulted in the successful production of a widely paraspecific antiserum against at least 36 neurotoxic venoms of 28 species encompassing 10 genera and from 20 countries on four continents, and possibly against all elapid venoms with α-neurotoxins as the lethal toxins. These results indicate that, with optimizations of the composition of the 'diverse toxin repertoire', the immunization scheme and antibody fractionation to increase the antivenom neutralizing potency, an effective universal antivenom against the neurotoxic elapid snakes of the world can be produced.

Potency Testing of Venoms and Antivenoms in Embryonated Eggs: An Ethical Alternative to Animal Testing.

Venoms are complex mixtures of biologically active molecules that impact multiple physiological systems. Manufacture of antivenoms (AVs) therefore requires potency testing using in vivo models to ensure AV efficacy. As part of ongoing research to replace small animals as the standard model for AV potency testing, we developed an alternate in vivo method using the embryonated egg model (EEM). In this model, the survival of chicken embryos envenomated in ovo is determined prior to 50% gestation, when they are recognized as animals by animal welfare legislation. Embryos were found to be susceptible to a range of snake, spider, and marine venoms. This included funnel-web spider venom for which the only other vertebrate, non-primate animal model is newborn mice. Neutralization of venom with standard AV allowed correlation of AV potency results from the EEM to results from animal assays. Our findings indicate that the EEM provides an alternative, insensate in vivo model for the assessment of AV potency. The EEM may enable reduction or replacement of the use of small animals, as longer-term research that enables the elimination of animal use in potency testing continues.

Wound Infections from Taiwan Cobra (Naja atra) Bites: Determining Bacteriology, Antibiotic Susceptibility, and the Use of Antibiotics-A Cobra BITE Study.

Wound necrosis and secondary infection are common complications after Naja atra bites. Clinical tools to evaluate the infection risk after Taiwan cobra bites are lacking. In this Cobra BITE study, we investigated the prevalence of wound infection, bacteriology, and corresponding antibiotic usage in patients presenting with Taiwan cobra snakebites. Patients with wound infection lacking tissue necrosis were included in developing Cobra BITE score utilizing univariate and multiple logistic regression, as patients with wound necrosis require antibiotics for infection treatment. 8,295,497 emergency department visits occurred in the span of this study, with 195 of those patients being diagnosed as having cobra bites. Of these patients, 23 had wound necrosis, and 30 had wound infection, resulting in a wound infection rate of 27.2% (53/195). Enterococcus faecalis and Morganella morganii were the main bacteria identified in the culture report regardless of whether patients' wounds had necrosis. As per our Cobra BITE score, the three factors predicting secondary wound infection after cobra bites are hospital admission, a white blood cell count (in 103/µL) × by neu-trophil-lymphocyte ratio value of ≥114.23, and the use of antivenin medication. The area under the receiver operating characteristic curve for the Cobra BITE score system was 0.88; ideal sensitivity and specificity were 0.89 and 0.76. This scoring system enables the assessment of wound infections after N. atra bites, and it could be modified and improved in the future for other Naja spp. bites.

Effect of Indian Polyvalent Antivenom in the Prevention and Reversal of Local Myotoxicity Induced by Common Cobra (Naja naja) Venom from Sri Lanka In Vitro.

Bites by many Asiatic and African cobras (Genus: Naja) cause severe local dermonecrosis and myonecrosis, resulting in permanent disabilities. We studied the time scale in which two Indian polyvalent antivenoms, VINS and Bharat, remain capable of preventing or reversing in vitro myotoxicity induced by common cobra (Naja naja) venom from Sri Lanka using the chick biventer cervicis nerve-muscle preparation. VINS fully prevented while Bharat partially prevented (both in manufacturer recommended concentrations) the myotoxicity induced by Naja naja venom (10 µg/mL) when added to the organ baths before the venom. However, both antivenoms were unable to reverse the myotoxicity when added to organ baths 5 and 20 min post-venom. In contrast, physical removal of the venom from the organ baths by washing the preparation 5 and 20 min after the venom resulted in full and partial prevention of the myotoxicity, respectively, indicating the lag period for irreversible cellular injury. This suggests that, although the antivenoms contain antibodies against cytotoxins of the Sri Lankan Naja naja venom, they are either unable to reach the target sites as efficiently as the cytotoxins, unable to bind efficiently with the toxins at the target sites, or the binding with the toxins simply fails to prevent the toxin-target interactions.

Population pharmacokinetics of Pseudechis porphyriacus (red-bellied black snake) venom in snakebite patients.

OBJECTIVES: Understanding the time course of venom exposure in snakebite patients is important for the optimisation of treatment including antivenom dose and timing. We aimed to investigate the pharmacokinetics of red-bellied black snake (RBBS; Pseudechis porphyriacus) venom in envenomed patients. METHODS: Timed venom concentration data were obtained from patients with RBBS envenomation recruited to the Australian Snakebite Project (ASP), including demographics and antivenom treatment. Venom concentrations were measured using an enzyme immunoassay. Data were modelled using NONMEM version 7.3. Uncertainty in venom "dose" was accounted for by arbitrarily fixing the average amount to 1 mg and incorporating between-subject variability on relative bioavailability. A scale parameter for venom clearance was implemented to account for the rapid venom clearance following antivenom dosing. A sensitivity analysis was performed to determine the magnitude of venom clearance amplification. RESULTS: There were 457 venom concentrations in 114 patients (median age 41, 2-90 y; 80 male). Antivenom was administered to 54 patients a median of 4.2 h post-bite (0.67 to 32 h). A one-compartment model with first-order absorption and elimination provided the best description of the data. The estimated clearance and volume of distribution were 5.21 L/h and 39.9 L, respectively. The calculated elimination half-life of P. porphyriacus venom from the final pharmacokinetic model was 5.35 ± 0.36 h. The variability in the relative dose of injected venom was 140%. Antivenom administration increased venom clearance by 40-fold. Ten patients showed evidence of a double peak in the absorption profile. CONCLUSION: The information on the exposure time of venom in the body following envenomation will help improve treatment and the timing of antivenom.

Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib.

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

In vitro anti-venom potential of various solvent based leaf extracts of Andrographis serpyllifolia (Rottler ex Vahl) Wight against Naja naja and Daboia russelli.

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.

Anticoagulant Micrurus venoms: Targets and neutralization.

Snakebite is a neglected tropical disease with a massive global burden of injury and death. The best current treatments, antivenoms, are plagued by a number of logistical issues that limit supply and access in remote or poor regions. We explore the anticoagulant properties of venoms from the genus Micrurus (coral snakes), which have been largely unstudied, as well as the effectiveness of antivenom and a small-molecule phospholipase inhibitor-varespladib-at counteracting these effects. Our in vitro results suggest that these venoms likely interfere with the formation or function of the prothrombinase complex. We find that the anticoagulant potency varies widely across the genus and is especially pronounced in M. laticollaris. This variation does not appear to correspond to previously described patterns regarding the relative expression of the three-finger toxin and phospholipase A2 (PLA2) toxin families within the venoms of this genus. The coral snake antivenom Coralmyn, is largely unable to ameliorate these effects except for M. ibiboboca. Varespladib on the other hand completely abolished the anticoagulant activity of every venom. This is consistent with the growing body of results showing that varespladib may be an effective treatment for a wide range of toxicity caused by PLA2 toxins from many different snake species. Varespladib is a particularly attractive candidate to help alleviate the burden of snakebite because it is an approved drug that possesses several logistical advantages over antivenom including temperature stability and oral availability.

In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity.

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10-15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

Development of a Treatment Protocol for Cobra (Naja naja) Bite Envenoming in Dogs.

There is limited information on clinical profiles, treatment, and management aspects of Indian cobra (Naja naja) bite envenoming in dogs in Sri Lanka. Dogs with cobra bites presented to the Veterinary Teaching Hospital (VTH), University of Peradeniya, were prospectively studied over a period of 72 months; local and systemic clinical manifestations and hematological abnormalities were recorded. We studied 116 cobra bite envenomings in dogs. A grading system was established using a combination of anatomical site of fang marks, as well as local and systemic clinical manifestations. Accordingly, treatment strategies were established using Indian polyvalent antivenom (AVS). Pain and swelling at the bite site were major clinical signs observed, while neurotoxic manifestations (mydriasis, wheezing, and crackles) were detected in most dogs. Leukocytosis was observed in 78% of them. Statistical analysis revealed that the grading scores obtained were compatible to initiate AVS administration according to the severity. The minimum number required was 2 AVS vials (range 2-12). Almost 20% of the dogs developed wheezing, crackles, hypersalivation, restlessness, and dyspnea as adverse reactions to AVS treatment. Necrotic wounds on bitten anatomical sites developed in 19% of the dogs and 2.5% developed acute kidney injuries as a consequence of envenoming crisis. Despite treatment, 3% of dogs died. No dry bites were recorded.

Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom.

OBJECTIVE: Naja ashei is a snake of medical importance in Kenya, Ethiopia, Somalia, Uganda, and Tanzania. Little is known about the enzymatic (snake venom phospholipases A2; svPLA2's) and toxic (lethal) activities of N. ashei venom and crucially, the safety and capacity of available antivenom to neutralize these effects. This study aimed to determine the enzymatic and toxic activities of N. ashei venom and the capacity of Indian and Mexican manufactured antivenoms to neutralize these effects. The protein content of the venom and the test antivenoms were also evaluated. A 12-point log concentration-response curve (0.5-22.5 µg/mL) was generated on an agarose-egg yolk model to predict the svPLA2 activity of the venom. The toxicity profiles of the venom and antivenoms were evaluated in the brine shrimp lethality assay. Lowry's method was used for protein estimation. RESULTS: Low and intermediate concentrations of the venom exhibited similar svPLA2 activities. The same was true for concentrations > 15 µg/mL. Intermediate and high doses of the venom exhibited similar mortalities in brine shrimp and test antivenoms were generally non-toxic but poorly neutralized svPLA2 activity. Mexican manufactured antivenom had lower protein content but neutralized venom-induced brine shrimp lethality much more effectively than Indian manufactured antivenom.

Clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in Australian elapid snake venoms.

Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the blood clotting factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. Cofactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species.

Anticoagulant activity of krait, coral snake, and cobra neurotoxic venoms with diverse proteomes are inhibited by carbon monoxide.

BACKGROUND: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. METHODS: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. RESULTS: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. CONCLUSION: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Proteomics of Naja kaouthia venom from North East India and assessment of Indian polyvalent antivenom by third generation antivenomics.

In the present study, venom composition, toxic effects, and immunological characteristics of Naja kaouthia venom from North East India has been studied. Using RP-HPLC, venom components were separated and proteins in the fractions were identified using ESI-LC MS/MS. Proteins identified belong to 9 different snake venom protein families. Three finger toxins and PLA2 were the most abundant protein families detected by mass spectrometry analysis. The other minor proteins families identified in the venom were kunitz-type serine inhibitors, waprin, L-amino acid oxidase, CRISP, vespyrn, nerve growth factor and metalloproteinase. This proteome composition correlated with the tested enzymatic and toxic activities of the venom. Western blot and third generation antivenomics analysis using Vins polyvalent antivenom revealed immunoreactivity towards Naja kaouthia venom of North East India. Concentration-dependent immunocapturing profile carried out using RP-HPLC displayed immunerecognition of majority of venom proteins of Naja kaouthia except few three-finger toxins. Presence of such non-immunodepleted toxins apparently may affect the performance of Vins polyvalent antivenom. Thus, inclusion of antibodies of most relevant non-immunorecognized toxins in antivenom might help to improve the quality of antivenom. BIOLOGICAL SIGNIFICANCE: Envenomings by genus Naja, represent a serious medical problem in Asian countries including North east India. In North East India, Naja kaouthia is most prevalent cobra species causing a large number of fatalities. To gain deeper insight into the spectrum of medically relevant toxins, we applied proteomics approach to unveil the proteome profile of Naja kaouthia venom. The proteomic analysis divulged the presence of two major protein families: three finger toxins and phospholipases A2. In general, polyvalent antivenom is administered for Naja kaouthia envenomings, however, this venom is not included in the immunization mixtures (only Indian Big Four venoms) for production of these polyvalent antivenoms. For the first time, third generation antivenomics approach was used to decipher maximal binding capacity of Indian polyvalent antivenom against Naja kaouthia venom. Although Vins polyvalent antivenom was effective in immunocapturing majority of venom components, however, large amount of antivenom was required to immunocapture the venom proteins. Moreover, the study revealed poor immunorecognition capacity of Vins antivenom towards four three finger toxin subtypes. This may have significant impact on antivenom efficacy in treating Naja kaouthia envenomings.

First look into the venom of Roatan Island's critically endangered coral snake Micrurus ruatanus: Proteomic characterization, toxicity, immunorecognition and neutralization by an antivenom.

A proteomic and toxicological study of the venom from one specimen of Micrurus ruatanus, a critically endangered coral snake species endemic to Roatan Island, Honduras, was carried out. Immunorecognition and neutralization of venom lethality by an anticoral antivenom was also evaluated. Forty peaks were collected from RP-HPLC fractionation of the venom. After SDS-PAGE analysis, fifty-eight bands were examined by MALDI-TOF/TOF mass spectrometry. Micrurus ruatanus venom displayed a three-finger toxin (3FTx)-rich venom phenotype, as well as a significant amount of phospholipases A2 (PLA2s). Various other proteins were identified, including Kunitz-type inhibitor proteins, L-amino acid oxidases, C-type lectin/lectin-like, metalloproteinases, serine proteinases, vespryn/ohanin, 5'-nucleotidases, glutathione peroxidases, and phosphodiesterases. Micrurus ruatanus venom displayed significant PLA2 activity in vitro and myotoxicity in vivo. The venom showed high lethal potency in mice, being one of the most lethal in Central America. The anticoral antivenom (SAC-ICP) produced by Instituto Clodomiro Picado neutralized the lethal activity of the venom. Major fractions with relevant lethal activity were also identified by a screening analysis. SIGNIFICANCE: The proteomic characterization, toxicity, immunorecognition and neutralization of Micrurus ruatanus venom have been determined for the first time. This coral snake is endemic to Roatan Island and contains a three-finger toxin-rich venom that displayed a potent lethal activity in mice. The anticoral antivenom produced by Instituto Clodomiro Picado neutralized the lethal activity of this venom in vivo, and therefore should be effective in the treatment of envenomings by this snake.

Neutralizing properties of LY315920 toward snake venom group I and II myotoxic phospholipases A2.

A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.

Human Monoclonal scFvs that Neutralize Fribrinogenolytic Activity of Kaouthiagin, a Zinc-Metalloproteinase in Cobra (Naja kaouthia) Venom.

Snake venom-metalloproteinases (SVMPs) are the primary factors that disturb hemostasis and cause hemorrhage in the venomous snake bitten subjects. Kaouthiagin is a unique SVMP that binds and cleaves von Willebrand factor (vWF) at a specific peptide bond leading to inhibition of platelet aggregation, which enhances the hemorrhage. Kaouthiagin is a low abundant venom component of Thai cobra (Naja kaouthia); thus, most horse-derived antivenins used for cobra bite treatment do not contain adequate anti-kaouthiagin. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with kaouthiagin activity for further clinical use. Kaouthiagin was purified from N. kaouthia-holovenom by a single-step gel-filtration chromatography. The purified venom component was used in phage-biopanning to select the kaouthiagin-bound HuscFv-displayed-phage clones from a HuscFv-phage display library. The selected phages were used to infect Escherichia coli bacteria. Soluble HuscFvs expressed by three phage-transformed-E. coli clones interfered with cobra kaouthiagin binding to human vWF. Computerized simulation indicated that HuscFv of two phage-transformed E. coli clones formed contact interface with kaouthiagin residues at or near catalytic site and effectively inhibited fibrinogenolytic activity of the kaouthiagin. The HuscFvs have therapeutic potential as an adjunct of antivenins in treatment of bleeding caused by venomous snakebites.