Toxinological and pharmacological characterization of Southeast Asian Naja kaouthia (Monocled cobra) venom /Tan Kae Yi

Snakebite envenomation is a neglected tropical disease and a serious public health problem in many countries in the tropics and subtropics, including Malaysia and Thailand. Antivenom remains as the only definitive treatment for snakebite envenomation; unfortunately, many nations do not have financial and technical resources to produce their own specific snake antivenom. These nations are relying on imported antivenoms that may not be very effective in treating envenomation by local snake species, due to geographical variations in the venom composition. These differences are medically relevant as they can lead to varied envenoming effects and treatment outcome. The monocled cobra (Naja kaouthia) is one of the most common dangerous species widely distributed in Indochina, northern Malayan Peninsula, northeastern India and southern China. The N. kaouthia venom from Thailand and Malaysia were previously shown to be substantially different in their median lethal doses (LD50); however, the differences in their venom compositions and pharmacological actions have not been elucidated. This present work profiled the venom proteomes of N. kaouthia from three different geographical regions, i.e. Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V) using reverse-phase HPLC, SDS-PAGE and tandem mass spectrometry. The venom lethality and mechanism of neuromuscular blockade were also investigated in vivo using mice and in vitro using chick biventer cervicis nerve-muscle (CBCNM) preparation, while the neutralization of venom-induced toxic effects was assessed using Thai N. kaouthia Monovalent Antivenom (NKMAV) and/or Neuro Polyvalent Antivenom (NPAV) produced by Thai Red Cross Society. The venom proteome results revealed remarkable biogeographical variations in all three N. kaouthia venoms, with three-finger toxin (3FTx) being the most abundant but also the most varied among three venom samples studied. These venoms also exhibit differences in venom lethality and neuromuscular depressant activity that reflect the proteomic findings, with NK-T being the most lethal and most neurotoxic. Despite the variation in proteome, Thai-produced antivenoms were capable of neutralizing toxic effects of all three venoms with varying degree of effectiveness. The findings suggest that Thai-produced antivenoms could be used for the treatment of N. kaouthia bites in Malaysia and Vietnam. However, the recommended antivenom dosage may be tailored and further optimized. This present work also investigated the toxin-specific neutralization by NKMAV to understand why cobra antivenoms are generally of limited neutralization potency (< 2 mg/ml). The principal toxins of NK-T and Malaysian beaked sea snake (Hydrophis schistosus, HS-M) were purified and their neutralization by NKMAV and Australian CSL Sea Snake Antivenom (SSAV) were investigated. The neutralization profiles showed the low efficacy of antivenoms against low molecular mass toxins, particularly against the short neurotoxin (SNTX) of both venoms examined. This indicates that the limiting factor in neutralization potency is the poor ability of antivenoms to neutralize SNTX. Nevertheless, the SSAV was still substantially superior to NKMAV in neutralizing SNTX, presumably because the sea snake venom used as an immunogen in SSAV production contains a large amount of SNTX. The toxin-specific neutralization findings suggest that it is possible to improve the efficacy of cobra antivenom by increasing the amount of SNTX in the immunogen

<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

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

Fig.9_LAAO_Jalview

Primary multiple sequence alignment of Fig.9 (LAAO) performed with MUSCLE program using Jalview software

Data from: Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah)

Background: The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS. Results: Transcriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A 2 (PLA 2 s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). Seventeen toxin families exhibited low mRNA expression, including hyaluronidase, DPP-IV and 5’-nucleotidase that were not previously reported in the venom-gland transcriptome of a Balinese O. hannah. On the other hand, the MOh proteome includes 3FTxs, the most abundantly expressed proteins in the venom (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 non-conventional 3FTx. Neurotoxins comprise the major 3FTxs in the MOh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. The presence of toxic enzymes such as LAAOs, SVMPs and PLA 2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) are in agreement with the poor cross-neutralization activity of N. kaouthia antivenom used against MOh venom. Besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5’-nucleotidase, and DPP-IV in the venom proteome suggests its probable hypotensive action in subduing prey. Conclusion: This study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). The results correlate with the pathophysiological actions of MOh venom, and dispute the use of Naja cobra antivenoms to treat MOh envenomation. The findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom

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 &lt; Thai monocled cobra monovalent antivenom &lt; 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

Fig.5a_3FTx_All_Jalview

Primary multiple sequence alignment of Fig.5a (All MOh 3FTxs) performed with MUSCLE program using Jalview software

Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper:Unveiling a deeply conserved atypical toxin arsenal

Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD(50)) (0.56–0.63 μg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A(2), serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3–5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes