Antivenom Cross-Neutralization of the Venoms of Hydrophis schistosus and Hydrophis curtus, Two Common Sea Snakes in Malaysian Waters

Sea snake envenomation is a serious occupational hazard in tropical waters. In Malaysia, the beaked sea snake (Hydrophis schistosus, formerly known as Enhydrina schistosa) and the spine-bellied sea snake (Hydrophis curtus, formerly known as Lapemis curtus or Lapemis hardwickii) are two commonly encountered species. Australian CSL sea snake antivenom is the definitive treatment for sea snake envenomation; it is unfortunately extremely costly locally and is not widely available or adequately stocked in local hospitals. This study investigated the cross-neutralizing potential of three regionally produced anti-cobra antivenoms against the venoms of Malaysian H. schistosus and H. curtus. All three antivenoms conferred paraspecific protection from sea snake venom lethality in mice, with potency increasing in the following order: Taiwan bivalent antivenom < Thai monocled cobra monovalent antivenom < Thai neuro polyvalent antivenom (NPAV). NPAV demonstrated cross-neutralizing potencies of 0.4 mg/vial for H. schistosus venom and 0.8 mg/vial for H. curtus, which translates to a dose of less than 20 vials of NPAV to neutralize an average amount of sea snake venom per bite (inferred from venom milking). The cross-neutralization activity was supported by ELISA cross-reactivity between NPAV and the venoms of H. schistosus (58.4%) and H. curtus (70.4%). These findings revealed the potential of NPAV as a second-line treatment for sea snake envenomation in the region. Further profiling of the cross-neutralization activity should address the antivenomic basis using purified toxin-based assays

Evaluation of the geographical utility of Eastern Russell's viper (Daboia siamensis) antivenom from Thailand and an assessment of its protective effects against venom-induced nephrotoxicity.

BACKGROUND Daboia siamensis (Eastern Russell's viper) is a medically important snake species found widely distributed across Southeast Asia. Envenomings by this species can result in systemic coagulopathy, local tissue injury and/or renal failure. While administration of specific antivenom is an effective treatment for Russell's viper envenomings, the availability of, and access to, geographically-appropriate antivenom remains problematic in many rural areas. In this study, we determined the binding and neutralizing capability of antivenoms manufactured by the Thai Red Cross in Thailand against D. siamensis venoms from four geographical locales: Myanmar, Taiwan, China and Thailand. METHODOLOGY/PRINCIPLE FINDINGS The D. siamensis monovalent antivenom displayed extensive recognition and binding to proteins found in D. siamensis venom, irrespective of the geographical origin of those venoms. Similar immunological characteristics were observed with the Hemato Polyvalent antivenom, which also uses D. siamensis venom as an immunogen, but binding levels were dramatically reduced when using comparator monovalent antivenoms manufactured against different snake species. A similar pattern was observed when investigating neutralization of coagulopathy, with the procoagulant action of all four geographical venom variants neutralized by both the D. siamensis monovalent and the Hemato Polyvalent antivenoms, while the comparator monovalent antivenoms were ineffective. These in vitro findings translated into therapeutic efficacy in vivo, as the D. siamensis monovalent antivenom was found to effectively protect against the lethal effects of all four geographical venom variants preclinically. Assessments of in vivo nephrotoxicity revealed that D. siamensis venom (700 μg/kg) significantly increased plasma creatinine and blood urea nitrogen levels in anaesthetised rats. The intravenous administration of D. siamensis monovalent antivenom at three times higher than the recommended scaled therapeutic dose, prior to and 1 h after the injection of venom, resulted in reduced levels of markers of nephrotoxicity and prevented renal morphological changes, although lower doses had no therapeutic effect. CONCLUSIONS/SIGNIFICANCE This study highlights the potential broad geographical utility of the Thai D. siamensis monovalent antivenom for treating envenomings by the Eastern Russell's viper. However, only the early delivery of high antivenom doses appears to be capable of preventing venom-induced nephrotoxicity

Health promotion activity in Kampung Rangalau , Kota Belud

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Toxinological characterizations of the venom of hump-nosed pit viper (Hypnale hypnale) / Tan Choo Hock

Hump-nosed pit viper (Hypnale hypnale) is a medically important snake in Sri Lanka and Western Ghats of India. Envenomation by this snake still lacks effective antivenom clinically. The species is also often misidentified, resulting in inappropriate treatment. The median lethal dose (LD50) of H. hypnale venom varies from 0.9 μg/g intravenously to 13.7 μg/g intramuscularly in mice. The venom shows procoagulant, hemorrhagic, necrotic, and various enzymatic activities including those of proteases, phospholipases A2 and L-amino acid oxidases which have been partially purified. The monovalent Malayan pit viper antivenom and Hemato polyvalent antivenom (HPA) from Thailand effectively cross-neutralized the venom’s lethality in vitro (median effective dose, ED50 = 0.89 and 1.52 mg venom/mL antivenom, respectively) and in vivo in mice, besides the procoagulant, hemorrhagic and necrotic effects. HPA also prevented acute kidney injury in mice following experimental envenomation. Therefore, HPA may be beneficial in the treatment of H. hypnale envenomation. H. hypnale-specific antiserum and IgG, produced from immunization in rabbits, effectively neutralized the venom’s lethality and various toxicities, indicating the feasibility to produce an effective specific antivenom with a common immunization regime. On indirect ELISA, the IgG cross-reacted extensively with Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), suggesting that the two phylogenically related snakes share similar venoms antigenic properties. Doublesandwich ELISA was specific and able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common Sri Lankan viperids) in human sera; hence it may be useful in diagnostics and venom level monitoring especially during clinical studies. In rabbits, the venom when injected intravenously showed a rapid distribution phase (t1/2α = 0.6 h) and a slow elimination phase (t1/2β = 20 h), consistent with prolonged abnormal hemostasis reported. The intramuscular bioavailability was exceptionally low (Fi.m = 4%), accountable for the highly varied LD50 between intravenous and intramuscular envenomings in animals. HPA infused post-envenomation markedly reduced the serum venom levels and subsequently induced venom redistribution. The redistributed venom was completely neutralized by a second dose of HPA. Proteomic study by shotgun-liquid chromatography-mass spectrometry/mass spectrometry (shotgun-LC-MS/MS) revealed 52 proteins in the venom, 70% of which are toxinologically related. The combined use of reverse-phase high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide sequencing and mass fingerprinting technologies enabled the identification of the venom major components, i.e. zinc-dependent metalloproteases, phospholipases A2, Lamino acid oxidases, serine proteases and C-type lectins. These toxins correlate with the venom’s principal effects: hematoxicity (hemorrhage, coagulopathy) and tissue destruction (necrosis). Understanding of the venom composition is essential for ascertaining the principal toxins, and for optimizing antivenom formulation. Furthermore, hypnobin, a 37 kDa thrombin-like enzyme was purified. It exhibits arginine esterese and amidase activities, besides distinct specificity towards different mammalian fibrinogens. In fibrinogen coagulation, hypnobin predominantly releases fibrinopeptide A, followed slowly by a small amount of fibrinopeptide B. The findings provide insights into the structures and mechanism of the thrombin-like enzyme, which are important for elucidation of the pathophysiology and for potential drug discovery, e.g. an anticoagulant for thromboembolic disorders

De Novo Venom-Gland Transcriptomics of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia—Next-Generation Sequencing, Functional Annotation and Toxinological Correlation

Envenomation resulted from sea snake bite is a highly lethal health hazard in Southeast Asia. Although commonly caused by sea snakes of Hydrophiinae, each species is evolutionarily distinct and thus, unveiling the toxin gene diversity within individual species is important. Applying next-generation sequencing, this study investigated the venom-gland transcriptome of Hydrophis curtus (spine-bellied sea snake) from Penang, West Malaysia. The transcriptome was de novo assembled, followed by gene annotation and sequence analyses. Transcripts with toxin annotation were only 96 in number but highly expressed, constituting 48.18% of total FPKM in the overall transcriptome. Of the 21 toxin families, three-finger toxins (3FTX) were the most abundantly expressed and functionally diverse, followed by phospholipases A2. Lh_FTX001 (short neurotoxin) and Lh_FTX013 (long neurotoxin) were the most dominant 3FTXs expressed, consistent with the pathophysiology of envenomation. Lh_FTX001 and Lh_FTX013 were variable in amino acid compositions and predicted epitopes, while Lh_FTX001 showed high sequence similarity with the short neurotoxin from Hydrophis schistosus, supporting cross-neutralization effect of Sea Snake Antivenom. Other toxins of low gene expression, for example, snake venom metalloproteinases and L-amino acid oxidases not commonly studied in sea snake venom were also identified, enriching the knowledgebase of sea snake toxins for future study

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

Snake venom proteomics, immunoreactivity and toxicity neutralization studies for the asiatic mountain pit vipers, ovophis convictus, ovophis tonkinensis, and hime habu, ovophis okinavensis

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40–60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions

<i>De Novo</i> Assembly of Venom Gland Transcriptome of <i>Tropidolaemus wagleri</i> (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