Lawan Chanhome

Identification of alpha-bungarotoxin (A31) as the major postsynaptic neurotoxin, and complete nucleotide identity of a genomic DNA of Bungarus candidus from Java with exons of the Bungarus multicinctus alpha-bungarotoxin (A31) gene.

The Malayan krait (Bungarus candidus) is one of the most medically significant snake species in Southeast Asia. No specific antivenom exists to treat envenoming by this species. Death within 30 min after its bite has been reported from Java, suggesting the presence of highly lethal postsynaptic neurotoxins in the venom of these snakes. We purified and identified the major postsynaptic toxin in the venom of B. candidus from Java. The toxin was indistinguishable from alpha-bungarotoxin (A31), a toxin originally isolated from Bungarus multicinctus, in its mass (7983.75 Da), LD50 (0.23 microg/g in mice i.p.), affinity to nicotinic acetylcholine receptors, and by its 40 N-terminal amino acid residues as determined by Edman degradation. Identity with alpha-bungarotoxin was confirmed by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of alpha-bungarotoxin (A31) and part of its signal peptide, revealing complete identity to the alpha-bungarotoxin (A31) gene in exon and 98.9% identity in intron sequences. The entire mitochondrial cytochrome b gene of the krait species B. candidus from Java and B. multicinctus from Taiwan was sequenced for comparison, suggesting that these snakes are phylogenetically closely related. alpha-Bungarotoxin appears to be widely present and conserved in Southeast and East Asian black-and-white kraits across populations and taxa.

Venomous snake husbandry in Thailand

A captive breeding program for venomous Thai snakes was established at the Queen Saovabha Memorial Institute at Bangkok, Thailand. This was necessary to secure a stable, healthy, and species-confirmed source of snake venom for antivenom production. In 1994, wild-caught specimens were collected, sexed, quarantined, and housed appropriately. All data in this report, with the exclusion of Table 6, were collected from 1994 to 1997. Two species were bred successfully in captivity to date during this study period. Although captive breeding has not yet been achieved with all species and subspecies, our early success was encouraging.

Comparative venom gland transcriptomics of Naja kaouthia (monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

Background The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology. Methods The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T. Results and discussion The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

Cross-neutralization of Thai cobra (Naja kaouthia) and spitting cobra (Naja siamensis) venoms by Thai cobra antivenom

The neutralizing capacity of antivenom prepared against Thai cobra (Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra (Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 LD50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.

Characterization of venomous snakes of Thailand

Background: Envenoming by snakebite is an important public health problem in rural tropics. Venomous snake families such as Elapidae and Viperidae frequently produce severe poisoning. Anti-venoms are not available for all venomous snakes of Thailand and there is need for more development in this field. Objective: We characterized the important venomous snakes’ distribution of Thailand. Method: Venomous snake species are described in details including their identification, range, and extraterritorial distribution. Result: Eighteen snake species of the family Elapidae are summarized in their characteristics and distribution. There are three species of Naja, one species of Ophiophagus, three species of Bungarus, four species of Calliophis, one species of Sinomicrurus, two species of Laticauda, and four species of subfamily Hydrophiinae. Fifteen snake species of the family Viperidae consisting of one species of subfamily Viperinae and fourteen species of subfamily Crotalinae are also discussed. Conclusion: All these snakes are venomous and their venom is potentially fatal since birth.

A hemorrhagin as a metalloprotease in the venom of Trimeresurus purpureomaculatus: purification and characterization

A major hemorrhagin was purified from the venom of the Thai green pit viper (Trimeresurus purpureomaculatus) by gel filtration, ion-exchange and affinity chromatography. A 15-fold purification was achieved with an overall yield of 7% of hemorrhagic activity. The hemorrhagin was homogeneous according to disc- and SDS-PAGE as well as on immunodiffusion. The molecular weight determined by SDS-PAGE was 72kDa. The purified hemorrhagin expresses proteolytic activity with heat-denatured casein and hide powder azure, but it was free of AE-hydrolase and phospholipase activities. Both hemorrhagic and proteolytic activities were inhibited by EDTA, suggesting that the hemorrhagin is a metalloprotease. The hemorrhagin hydrolyzed all gelatin preparations derived from types I, II, III and IV collagen, whereas it hydrolyzed only type IV native collagen. The hemorrhagic activity was neutralized by Thai green pit viper antivenom raised to Trimeresurus albolabris venom.

Envenomation of mice by Thai Cobra (Naja kaouthia) venom: tolerable venom concentration and exposure time

Naja kaouthia venom appeared in circulation rapidly after intramuscular injection into mice. The venom concentration attained a maximum level with all doses examined after 20 min. The half value of the maximum level was obtained 1 min after injection when a dose of 4LD50 was used. A critical venom concentration endangering mice was assessed from venom concentration in the sera of mice envenomed with sublethal dose (LD50). A fatal condition was produced within 30 min at a venom concentration of 200-300 ng/ml or within 50 min at a venom concentration of 100-150 ng/ml.

Isolation of the major lethal toxin in the venom of Bungarus flaviceps

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.

Development of a polymerase chain reaction to distinguish monocellate cobra (Naja khouthia) bites from other common Thai snake species, using both venom extracts and bite-site swabs

A PCR technique was used in this study to identify and distinguish monocellate cobra snake bites using snake venoms and swab specimens from snake bite-sites in mice from bites by other common Thai snakes. The sequences of nucleotide primers were selected for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) since the sequences of monocellate cobra (Naja kaouthia) venom are still unknown. However, the 113-bp fragment of cDNA of the cobrotoxin-encoding gene was detected in the monocellate cobra venom using RT-PCR. This gene was not found in the venoms of Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), Daboia russelii siamensis (Siamese Russells Viper, and Calloselasma rhodostoma (Malayan pit viper). Moreover, direct PCR could detect a 665-bp fragment of the cobrotoxin-encoding gene in the monocellate cobra venom but not the other snake venoms. Likewise, this gene was only observed in swab specimens from cobra snake bite-sites in mice. This is the first report demonstrating the ability of PCR to detect the cobrotoxin-encoding gene from snake venoms and swab specimens. Further studies are required for identification of this and other snakes from the bite-sites on human skin.

Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes

Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion–deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.