Search Results (20)

Clinical symptoms of monocled cobra (Naja kaouthia) envenoming include the paralysis of extraocular muscles, local tissue necrosis and death through respiratory failure. These neurotoxic outcomes are mainly due to the inhibitory action of postsynaptic neurotoxins to nicotinic acetylcholine receptors. However, injuries involving respiratory muscles have rarely been investigated. In this study, we determined the effect of N. kaouthia envenoming on morphological changes in the rat diaphragm. The efficacy of cobra monovalent antivenom in neutralising the histopathological effects of N. kaouthia venom was also evaluated. The intramuscular (i.m.) administration of N. kaouthia venom (2 mg/kg) caused skeletal muscle fibre atrophy and ruptures of myofibrils shown via a light microscope study. Transmission electron microscopy (TEM) revealed the zig-zagging of the Z-band, mitochondrial damages and degeneration of the synaptic fold of the neuromuscular junction following experimental cobra envenoming for 4 h. Intravenous administration of cobra antivenom at manufacturer-recommended doses diminished histopathological changes in the diaphragm following the administration of cobra venom. The expression of NF-kB and MuRF1 in the experimentally N. kaouthia-envenomed diaphragm indicated inflammation and tissue atrophy in the immunofluorescence analysis, respectively. In this study, we found that there were respiratory muscle injuries following N. kaouthia envenoming. The early administration of monovalent N. kaouthia antivenom is capable of neutralising neurotoxic outcomes following cobra envenoming. Full article

Animal venoms are intricate and teem with potential for groundbreaking medical advancements. Although traditional methods for purifying venom proteins are effective, they usually require complicated, multi-step processes that lead to lower yields. Our study introduces an efficient, one-step technique for extracting venom-derived proteins through reverse-phase high-performance liquid chromatography (RP-HPLC). We carefully optimized the RP-HPLC process, focusing on the gradient elution conditions and the strategic use of our columns’ stationary phase characteristics, to enhance the effectiveness of our separations. This enabled us to efficiently isolate six venom proteins: melittin (2.846 kDa) from Apis mellifera with a yield of 4.5% and homogeneity of 99%; α-cobratoxin (7.821 kDa) from Naja kaouthia with a yield of 15% and homogeneity of 99%; α-bungarotoxin (7.983 kDa) from Bungarus multicinctus with a yield of 7% and purity of 99%; calciseptine (7.035 kDa) from Dendroaspis polylepis with a yield of 6% and homogeneity of 95%; notexin (13.593 kDa) from Notechis scutatus with a yield of 10% and homogeneity of 95%; and CVFm (150 kDa) from Naja melanoleuca with a yield of 0.8% and homogeneity of 94%. These were all accomplished in one step. This breakthrough simplifies the purification of venom peptides and proteins, making the process more feasible and economical. It paves the way for developing new drugs and promising treatments that are both more effective and precisely targeted. Full article

Snakes contain three types of phospholipase A₂ (PLA₂)-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA₂. PLIβ is the snake ortholog of leucine-rich α₂ glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA₂ and weak inhibitory activity against Gloydius brevicauda basic PLA₂. Elaphe climacophora PLIβ had weaker inhibitory activity against G. brevicauda basic PLA₂ than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA₂. Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA₂, PL-X, or G. brevicauda basic PLA₂. Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA₂, but, whether it actually functions as a PLA₂-inhibitory protein, PLIβ, depends on the snake. Full article

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins. Full article

This study investigated the clinical characteristics, treatments, and outcomes of envenomation involving cobra species in Thailand (Naja kaouthia, Naja siamensis, and Naja sumatrana). Data of patients who had been bitten by a cobra or inoculated via the eyes/skin in 2018–2021 were obtained from the Ramathibodi Poison Center. There were 1045 patients admitted during the 4-year study period (bite, n = 539; ocular/dermal inoculation, n = 506). Almost all patients with ocular/dermal inoculation had eye involvement and ocular injuries, but none had neurological effects. Most of the patients bitten by a cobra had local effects (69.0%) and neurological signs and symptoms (55.7%). The median interval between the bite and the onset of neurological symptoms was 1 h (range, 10 min to 24 h). Accordingly, patients should be observed closely in hospitals for at least 24 h after a bite. Intubation with ventilator support was required in 45.5% of patients and for a median duration of 1.1 days. Antivenom was administered in 63.5% of cases. There were nine deaths, most of which resulted from severe infection. Neurological effects and intubation were significantly more common after a monocled cobra bite than after a spitting cobra bite. The administration of antivenom with good supportive care, including the appropriate management of complications, especially wound infection, might decrease fatality. Full article

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA₂ activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA₂s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable. Full article

Taxonomic frameworks for medically important species such as cobras (genus Naja Laurenti, 1768; Squamata, Elapidae) are essential for the medical treatment of snake bites and accurate antivenin development. In this paper, we described the former N. kaouthia populations recorded from China as a new species and designated a neotype for N. atra-based morphological and mitochondrial phylogenetic analysis. The new species N. fuxisp. nov. was morphologically diagnosed from N. kaouthia by (1) regular single narrow crossband present on the middle and posterior parts of the dorsum (3–15, 7.9 ± 2.7, n = 32) and the dorsal surface of the tail (1–6, 4.2 ± 1.1, n = 32) of both adults and juveniles, buff-colored with dark fringes on both edges, vs. South Asian populations (n = 39) and Southeast Asian populations (n = 35) without cross bands, with irregular cross bands or multiple light-colored crossbands pairs, or densely woven lines; (2) small scales between the posterior chin shields, usually three (40%) or two (37%), rarely four (13%), or one (10%) (n = 30) vs. mostly one (81%) and rarely two (19%) (n = 28); (3) ventrals 179–205 (195.4 ± 6.7, n = 33) vs. South Asian populations 179–199 (188.7 ± 5.9, n = 12); Southeast Asian populations 168–186 (177.8 ± 4.9, n = 18). Phylogenetically, the new species forms an independent sister clade to the clade including N. atra, N. kaouthia, N. oxiana and N. sagittifera. Furthermore, the subspecies N. naja polyocellata should be resurrected and recognized as a full species, N. polyocellatacomb. nov., and the subspecies N. sumatrana miolepis should be resurrected. Full article

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. Full article

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. Full article

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve–muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms. Full article

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins (P01391), short-chain α-neurotoxins (P60770), and cardiotoxin A3 (P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries. Full article

The protein abundances of phospholipases A₂ in cobra venom proteomes appear to vary among cobra species. To determine the unique distribution of snake venom phospholipases A₂ (svPLA₂) in the cobras, the svPLA₂ activities for 15 cobra species were examined with an acidimetric and a colorimetric assay, using egg yolk suspension and 4-nitro-3-octanoyloxy benzoic acid (NOBA) as the substrate. The colorimetric assay showed significant correlation between svPLA₂ enzymatic activities with the svPLA₂ protein abundances in venoms. High svPLA₂ activities were observed in the venoms of Asiatic spitting cobras (Naja sputatrix, Naja sumatrana) and moderate activities in Asiatic non-spitters (Naja naja, Naja atra, Naja kaouthia), African spitters (subgenus Afronaja), and forest cobra (subgenus Boulengerina). African non-spitting cobras of subgenus Uraeus (Naja haje, Naja annulifera, Naja nivea, Naja senegalensis) showed exceptionally low svPLA₂ enzymatic activities. The negligible PLA₂ activity in Uraeus cobra venoms implies that PLA₂ may not be ubiquitous in all snake venoms. The svPLA₂ in cobra envenoming varies depending on the cobra species. This may potentially influence the efficacy of cobra antivenom in specific use for venom neutralization. Full article

Cancer is a deadly disease and there is an urgent need for the development of effective and safe therapeutic agents to treat it. Snake venom is a complex mixture of bioactive proteins that represents an attractive source of novel and naturally-derived anticancer agents. Malaysia is one of the world’s most biodiverse countries and is home to various venomous snake species, including cobras. Naja kaouthia, Naja sumatrana, and Ophiophagus hannah are three of the most common cobra species in Malaysia and are of medical importance. Over the past decades, snake venom has been identified as a potential source of therapeutic agents, including anti-cancer agents. This present review highlights the potential anticancer activity of the venom and purified venom protein of N. kaouthia, N. sumatrana, and O. hannah. In conclusion, this review highlights the important role of the venom from Malaysian cobras as an important resource that researchers can exploit to further investigate its potential in cancer treatment. Full article

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. Full article

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation. Full article