Immunoreactivity and neutralization efficacy of Pakistani Viper Antivenom (PVAV) against venoms of Saw-scaled Vipers (Echis carinatus subspp.) and Western Russell's Vipers (Daboia russelii) from the Indian subcontinent.

Snakebite envenoming (SBE) is a priority Neglected Tropical Disease listed by the World Health Organization. South Asia is heavily affected, and virtually all countries in the region import polyvalent antivenom products from India for clinical use. The imported antivenoms, however, have suboptimal effectiveness due to geographical venom variation. Recently, a domestic bivalent product, named Pakistani Viper Antivenom (PVAV) has been developed specifically for Pakistani vipers, Echis carinatus sochureki and Daboia russelii. As a bivalent viperid antivenom, it is unknown yet if PVAV exhibits higher immunological binding and neutralization activities against viper venoms from distant locales compared with polyvalent antivenoms manufactured in India. This study thus examined the preclinical efficacy of PVAV against venoms of Western Russell's Vipers and Saw-scaled Viper subspecies from selected locales in the Indian subcontinent. PVAV generally outperformed the commonly used VINS polyvalent antivenom (VPAV, manufactured in India) in binding toward venoms, and showed superior or comparable neutralization efficacy against the venom procoagulant and hemorrhagic effects of Saw-scaled Vipers as well as Russell's Vipers from Pakistan and Sri Lanka. Based on normalized potency values, PVAV is far more potent than VPAV in neutralizing the lethality of all viper venoms, except that of the Indian Russell's Viper. The study shows conserved antigenicity of toxins responsible for major toxicity across these viperid venoms, and suggests the feasible production of a viper-specific antivenom with higher potency and broader geographical utility for the region.

De Novo Assembly of Venom Gland Transcriptome of Tropidolaemus wagleri (Temple Pit Viper, Malaysia) and Insights into the Origin of Its Major Toxin, Waglerin.

The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species.

De Novo Venom Gland Transcriptome Assembly and Characterization for Calloselasma rhodostoma (Kuhl, 1824), the Malayan Pit Viper from Malaysia: Unravelling Toxin Gene Diversity in a Medically Important Basal Crotaline.

In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.

An immunoinformatic approach to assessing the immunogenic capacity of alpha-neurotoxins in elapid snake venoms.

Introduction: Most elapid snakes produce venoms that contain alpha-neurotoxins (α-NTXs), which are proteins that cause post-synaptic blockade and paralysis in snakebite envenoming. However, existing elapid antivenoms are known for their low potency in neutralizing the neurotoxic activity of α-NTXs, while the immunological basis has not been elucidated. Methods: In this study, a structure-based major histocompatibility complex II (MHCII) epitope predictor of horse (Equus caballus), complemented with DM-editing determinant screening algorithm was adopted to assess the immunogenicity of α-NTXs in the venoms of major Asiatic elapids (Naja kaouthia, Ophiophagus hannah, Laticauda colubrina, Hydrophis schistosus, Hydrophis curtus). Results: The scoring metric M2R, representing the relative immunogenic performance of respective α-NTXs, showed all α-NTXs have an overall low M2R of <0.3, and most of the predicted binders feature non-optimal P1 anchor residues. The M2R scores correlate strongly (R2 = 0.82) with the potency scores (p-score) generated based on the relative abundances of α-NTXs and the neutralization potency of commercial antivenoms. Discussion: The immunoinformatic analysis indicates that the inferior antigenicity of α-NTXs is not only due to their small molecular size but also the subpar immunogenicity affected by their amino acid composition. Structural modification with conjugation and synthetic epitope as immunogen may potentially enhance the immunogenicity for improved antivenom potency against α-NTXs of elapid snakes.

Proteomic Analysis, Immuno-Specificity and Neutralization Efficacy of Pakistani Viper Antivenom (PVAV), a Bivalent Anti-Viperid Antivenom Produced in Pakistan.

Snakebite envenoming is a neglected tropical disease prevalent in South Asia. In Pakistan, antivenoms are commonly imported from India despite the controversy over their effectiveness. To solve the problem, the locals have developed the Pakistani Viper Antivenom (PVAV), raised against Sochurek's Saw-scaled Viper (Echis carinatus sochureki) and Russell's Viper (Daboia russelii) of Pakistani origin. This study is set to evaluate the composition purity, immuno-specificity and neutralization efficacy of PVAV. Chromatographic and electrophoretic profiling coupled with proteomic mass spectrometry analysis showed PVAV containing high-purity immunoglobulin G with minimum impurities, notably the absence of serum albumin. PVAV is highly immuno-specific toward the venoms of the two vipers and Echis carinatus multisquamatus, which are indigenous to Pakistan. Its immunoreactivity, however, reduces toward the venoms of other Echis carinatus subspecies and D. russelii from South India as well as Sri Lanka. Meanwhile, its non-specific binding activities for the venoms of Hump-nosed Pit Vipers, Indian Cobras and kraits were extremely low. In the neutralization study, PVAV effectively mitigated the hemotoxic and lethal effects of the Pakistani viper venoms, tested in vitro and in vivo. Together, the findings suggest the potential utility of PVAV as a new domestic antivenom for the treatment of viperid envenoming in Pakistan.

Characterizing and applying immunoglobulins in snakebite diagnostics: A simple and rapid venom detection assay for four medically important snake species in Southeast Asia.

Snakebite envenoming is a medical emergency requiring urgent and specific treatment. Unfortunately, snakebite diagnostics are scarce, time-consuming and lacking specificity. Hence, this study aimed to develop a simple, quick and specific snakebite diagnostic assay using animal antibodies. Anti-venom horse immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY) were produced against the venoms of four major medically important snake species in Southeast Asia, i.e., the Monocled Cobra (Naja kaouthia), Malayan Krait (Bungarus candidus), Malayan Pit Viper (Calloselasma rhodostoma), and White-lipped Green Pit Viper (Trimeresurus albolabris). Different capture:detection configurations of double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were constructed using both immunoglobulins, and the horse IgG:IgG-HRP configuration was found to be most selective and sensitive in detecting the corresponding venoms. The method was further streamlined to develop a rapid immunodetection assay, which is able to produce a visual color change within 30 min for discrimination between different snake species. The study shows it is feasible to develop a simple, quick and specific immunodiagnostic assay using horse IgG, which can be derived directly from antisera prepared for antivenom production. The proof-of-concept indicates it is a sustainable and affordable approach in keeping with on-going antivenom manufacturing activities for specific species in the region.

Decomplexation proteomic analysis and purity assessment of a biologic for snakebite envenoming: Philippine Cobra Antivenom.

BACKGROUND: Philippine Cobra Antivenom (PCAV) is the only snake antivenom manufactured in the Philippines. It is used clinically to treat envenoming caused by the Philippine Spitting Cobra (Naja philippinensis). While PCAV is effective pharmacologically, it is crucial to ensure the safety profile of this biologic that is derived from animal plasma. METHODS: This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions. CONCLUSIONS: Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.

Experience of using expired lyophilized snake antivenom during a medical emergency situation in Lao People's Democratic Republic--A possible untapped resource to tackle antivenom shortage in Southeast Asia.

OBJECTIVES: To evaluate the safety and efficacy of expired lyophilized snake antivenom of Thai origin during a medical emergency in 2020/2021 in Lao People's Democratic Republic. METHODS: Observational case series of patients with potentially life-threatening envenoming who consented to the administration of expired antivenom between August 2020 and May 2022. RESULTS: A total of 31 patients received the expired antivenom. Malayan pit vipers (Calloselasma rhodostoma) were responsible for 26 (84%) cases and green pit vipers (Trimeresurus species) for two cases (6%). In three patients (10%) the responsible snake could not be identified. Of these, two presented with signs of neurotoxicity and one with coagulopathy. A total of 124 vials of expired antivenom were administered. Fifty-nine vials had expired 2-18 months earlier, 56 vials 19-36 months and nine vials 37-60 months before. Adverse effects of variable severity were observed in seven (23%) patients. All 31 patients fully recovered from systemic envenoming. CONCLUSIONS: Under closely controlled conditions and monitoring the use of expired snake antivenom proved to be effective and safe. Discarding this precious medication is an unnecessary waste, and it could be a valuable resource in ameliorating the current shortage of antivenom. Emergency use authorization granted by health authorities and preclinical testing of expired antivenoms could provide the support and legal basis for such an approach.

Snake Venomics and Antivenomics of Cape Cobra (Naja nivea) from South Africa: Insights into Venom Toxicity and Cross-Neutralization Activity.

Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A2 (PLA2) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.

Preclinical assessment of VPEAV, a new trivalent antivenom for elapid snakebite envenoming in the Philippines: Proteomics, immunoreactivity and toxicity neutralization.

Snakebite envenoming is an important neglected tropical disease. Antivenom supply, however, remains limited in many parts of the world. This study aimed to examine the protein composition, immunoreactivity and neutralization efficacy of a new antivenom product (VINS Philippine Elapid Antivenoms, VPEAV) developed for the treatment of snakebite envenoming caused by the Philippine Cobra (Naja philippinensis), Samar Cobra (Naja samarensis) and King Cobra (Ophiophagus hannah). Size-exclusion chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry showed that VPEAV consisted of F(ab)'2 (∼90% of total antivenom proteins) with minimal protein impurities. Indirect ELISA showed varying immunoreactivity of VPEAV toward the different venoms (EC50 = 4-16 µg/ml), indicating distinct venom antigenicity between the species. In mice, the neutralization potency of VPEAV against the King Cobra venom was moderate (potency, P = 2.6 mg/ml, defined as the amount of venom completely neutralized per unit volume of antivenom). The potency was significantly lower against the N. philippinensis and N. samarensis venoms (P = 0.18-0.30 mg/ml), implying a higher dose may be needed for effective neutralization of the Naja venoms. Together, the findings suggest the potential and limitation of VPEAV in neutralizing the venom toxicity of the three Philippine elapid snakes.

Equatorial Spitting Cobra (Naja sumatrana) from Malaysia (Negeri Sembilan and Penang), Southern Thailand, and Sumatra: Comparative Venom Proteomics, Immunoreactivity and Cross-Neutralization by Antivenom.

The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A2 (PLA2), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom's lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49-0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.

An immunodetection assay developed using cobra cytotoxin-specific antibodies: Potential diagnostics for cobra envenoming.

Cobra (Naja spp.) envenoming is a life-threatening medical emergency, and a correct diagnosis is crucial to initiating timely and appropriate antivenom treatment. However, snakebite diagnostics remain unavailable in Southeast Asia. This study, therefore, developed an immunodetection assay with a potential diagnostic application for cobra envenoming. The cytotoxin of Naja kaouthia (Thai Monocled Cobra) (Nk-CTX) was purified from its venom to produce CTX-specific antibodies in rabbits and chickens. A double-antibody sandwich enzyme-linked immunosorbent assay was developed using the purified anti-Nk-CTX antibodies (immunoglobulin G and immunoglobulin Y), and its selectivity, specificity, and sensitivity for the venoms of five major cobra species in Southeast Asia (N. kaouthia, Naja sumatrana, Naja sputatrix, Naja siamensis, and Naja philippinensis) were studied. The results showed the immunoassay discriminates cobra venoms from other species commonly implicated in snakebites in Southeast Asia, i.e., the Malayan Krait, Many-banded Krait, King Cobra, Eastern Russell's Viper, Malayan Pit Viper and White-lipped Pit Viper. The immunoassay has a high sensitivity for the five cobra venoms, with detection limits (LoD) ranging from 0.6 to 2.6 ng/ml. Together, the findings suggest the potential diagnostic application of the cytotoxin immunoassay for cobra envenoming. The immunoassay was found to exhibit high immunoreactivity toward ten Asiatic cobra venoms (absorbance > 1.5), in contrast to African cobra venoms with low immunoreactivity (absorbance < 0.9). Considering the varying CTX antigenicity between Asiatic and African cobras, the immunoassay for African cobras should utilize antibodies produced specifically from the cytotoxins of African cobra venoms.

On characterizing the Red-headed Krait (Bungarus flaviceps) venom: Decomplexation proteomics, immunoreactivity and toxicity cross-neutralization by hetero-specific antivenoms.

The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.

Sharp-nosed Pit Viper (Deinagkistrodon acutus) from Taiwan and China: A comparative study on venom toxicity and neutralization by two specific antivenoms across the Strait.

In East Asia, the Sharp-nosed Pit Viper (Deinagkistrodon acutus) is a medically important venomous snake in Taiwan and China, two geographical areas long separated by the Taiwan Strait. Yet, snake venom variation is little known between specimens found across the Strait. This study thus investigated the intra-species variation of D. acutus venoms from Taiwan (Da-Taiwan) and China (Da-China) in their profiles of gel electrophoresis, toxicity, immunoreactivity and neutralization effect by antivenom. Da-China venom exhibited higher procoagulant, hemorrhagic and lethal activities than Da-Taiwan venom, presumably attributed to the higher abundance of moderate-to-high molecular weight toxins (procoagulants and hemorrhagins) in the venom. The mono-specific antivenoms produced in Taiwan (DaMAV-Taiwan) and China (DaMAV-China) were immunoreactive toward both venoms, and were able to neutralize the venom toxicity to different extents. DaMAV-Taiwan was more efficacious in neutralizing the venom procoagulant and lethal effects, while DaMAV-China was more potent against hemorrhagic effect. The discrepancy in efficacy between the two antivenoms could be due to varying proportions of neutralizing antibodies in the respective products, influenced by techniques of antibody raising and purification. Further study is warranted to elucidate variation in the proteome and antigenicity of D. acutus venom between snakes from Taiwan and China.

Cytotoxicity of Venoms and Cytotoxins from Asiatic Cobras (Naja kaouthia, Naja sumatrana, Naja atra) and Neutralization by Antivenoms from Thailand, Vietnam, and Taiwan.

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.

Snake Venomics: Fundamentals, Recent Updates, and a Look to the Next Decade.

Venomic research, powered by techniques adapted from proteomics, transcriptomics, and genomics, seeks to unravel the diversity and complexity of venom through which knowledge can be applied in the treatment of envenoming, biodiscovery, and conservation. Snake venom proteomics is most extensively studied, but the methods varied widely, creating a massive amount of information which complicates data comparison and interpretation. Advancement in mass spectrometry technology, accompanied by growing databases and sophisticated bioinformatic tools, has overcome earlier limitations of protein identification. The progress, however, remains challenged by limited accessibility to samples, non-standardized quantitative methods, and biased interpretation of -omic data. Next-generation sequencing (NGS) technologies enable high-throughput venom-gland transcriptomics and genomics, complementing venom proteomics by providing deeper insights into the structural diversity, differential expression, regulation and functional interaction of the toxin genes. Venomic tissue sampling is, however, difficult due to strict regulations on wildlife use and transfer of biological materials in some countries. Limited resources for techniques and funding are among other pertinent issues that impede the progress of venomics, particularly in less developed regions and for neglected species. Genuine collaboration between international researchers, due recognition of regional experts by global organizations (e.g., WHO), and improved distribution of research support, should be embraced.

Novel Neurotoxic Activity in Calliophis intestinalis Venom.

In this work, we investigated the in vitro neurotoxicity of Calliophis intestinalis venom using chick biventer cervicis neuromuscular preparations and electrophysiological analysis of voltage-gated sodium (NaV) channels expressed in HEK293 cells. We found that the indirect twitches of the neuromuscular preparations decreased over time when exposed to venom. However, the responses of these preparations to the agonists acetylcholine, carbachol, and potassium chloride were not changed after incubation with the venom. Our electrophysiological experiments show that C. intestinalis venom acts as a NaV channel antagonist-the first known from a vertebrate venom-by decreasing the peak current of NaV1.4 channels without changing the kinetics of activation or inactivation. Our proteomic results accord with earlier analyses and find that the venom contains three-finger toxins, cysteine-rich secretory proteins, kunitz peptides, phospholipase A2s, snake venom metalloproteases, and vespryns. Some of the three-finger toxins are similar to the δ-elapitoxins from the venom of the closely related Calliophis bivirgatus. However, δ-elapitoxins act as NaV channel agonists in C. bivirgatus whereas C. intestinalis venom contains NaV channel antagonists. The toxins and mechanisms responsible for the neuromuscular symptoms remain unclear as does the identity of the NaV channel antagonists. These aspects of this unusual venom require further study.

Varespladib (LY315920) rescued mice from fatal neurotoxicity caused by venoms of five major Asiatic kraits (Bungarus spp.) in an experimental envenoming and rescue model.

The venoms of Asiatic kraits (Bungarus spp.) contain various neurotoxic phospholipases A2 (beta-bungarotoxins) which can irreversibly damage motor nerve terminals, resulting in rapidly fatal suffocation by respiratory muscle paralysis or oral airway obstruction. Hence, there is a need of adjunct therapy at the pre-hospital stage to prevent or delay the onset of neurotoxicity, so that antivenom can be given within golden hour before the envenoming becomes antivenom-resistant. This study investigated the efficacy of varespladib, a small molecule PLA2 (phospholipase A2) inhibitor, given as a bolus subcutaneously upon the onset of krait venom-induced paralysis in a mouse experimental envenoming and rescue model, where the severity of neurotoxicity was scored and the survival rate was monitored over 24 h. Varespladib at 10 mg/kg effectively alleviated the neurotoxicity of Bungarus sindanus, Bungarus multicinctus and Bungarus fasciatus venoms, and rescued all mice from venom-induced lethality (100% survival). Varespladib at this dose, however, only partially reduced the neurotoxicity of Bungarus caeruleus and Bungarus candidus venoms, while all challenged mice were dead by 23 h (B. caeruleus) and 12 h (B. candidus). An increased dose of varespladib at 20 mg/kg markedly abated the venom neurotoxicity past 8 h of envenoming, and protected the mice from venom lethality (B. caeruleus: 75% survival; B. candidus: 100% survival). The finding is consistent with previous studies which demonstrated varespladib's inhibitory effect against some snake venoms. The findings suggest varespladib could be repurposed as an emergency drug for prevention or rescue (if given early enough) from the acute, neurotoxic envenoming syndromes caused by various major krait species in Asia.