Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 µg/kg, i.m.) or 0.9% NaCl solution (50 µL, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 µg/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (100­0.2 µg/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 µg/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 µg/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom. Conclusions: This study found that Malayan krait venoms from both populations possess myotoxic, cytotoxic and nephrotoxic activities. These findings may aid in clinical diagnosis and treatment of envenomed patients in the future.(AU)

Abundance of sea kraits correlates with precipitation.

Recent studies have shown that sea kraits (Laticauda spp.)--amphibious sea snakes--dehydrate without a source of fresh water, drink only fresh water or very dilute brackish water, and have a spatial distribution of abundance that correlates with freshwater sites in Taiwan. The spatial distribution correlates with sites where there is a source of fresh water in addition to local precipitation. Here we report six years of longitudinal data on the abundance of sea kraits related to precipitation at sites where these snakes are normally abundant in the coastal waters of Lanyu (Orchid Island), Taiwan. The number of observed sea kraits varies from year-to-year and correlates positively with previous 6-mo cumulative rainfall, which serves as an inverse index of drought. Grouped data for snake counts indicate that mean abundance in wet years is nearly 3-fold greater than in dry years, and this difference is significant. These data corroborate previous findings and suggest that freshwater dependence influences the abundance or activity of sea kraits on both spatial and temporal scales. The increasing evidence for freshwater dependence in these and other marine species have important implications for the possible impact of climate change on sea snake distributions.

Hyponatraemia, rhabdomyolysis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in southern Viet Nam.

Between 1998 and 2007, 42 patients admitted to Choray hospital, Ho Chi Minh City, and to two hospitals in adjacent regions in southern Viet Nam brought the Malayan kraits (Bungarus candidus) that had been responsible for biting them. Half of the patients had been bitten while they were asleep. Fang marks and numbness were the only local features of the bites. Common signs of neurotoxic envenoming included bilateral ptosis, persistently dilated pupils, limb weakness, breathlessness, hypersalivation, dysphonia and dysphagia. Thirty patients (71.4%) required endotracheal intubation of whom all but one were mechanically ventilated. Fourteen patients (33.3%) developed hypertension, 13 (31.0%) shock, 31 (73.8%) hyponatraemia (plasma sodium concentration < 130 mEq/l) and 30 (71.4%) showed evidence of mild rhabdomyolysis (peak plasma creatine kinase concentration 1375 +/- 140 micro/l). None developed acute kidney injury. All the patients were treated with a new monospecific B. candidus antivenom. There were no fatalities. Hyponatraemia has been reported previously in victims of Chinese kraits (Bungarus multicinctus) in northern Viet Nam and rhabdomyolysis in patients envenomed by B. niger in Bangladesh. These features of envenoming pose new problems for the management of krait bite cases in South east Asia and should stimulate a search for the causative venom toxins.

Structure-function studies on inhibitory activity of Bungarus multicinctus protease inhibitor-like protein on matrix metalloprotease-2, and invasion and migration of human neuroblastoma SK-N-SH cells.

In view of the findings that several Kunitz-type protein inhibitors suppress tumor invasion and metastasis, the aim of the present study is to explore whether Bungarus multicinctus protease inhibitor-like protein-2 (PILP-2) and PILP-3 exhibit anti-tumor activity. Although approximately 28% of amino acid substitutions occurred between PILP-2 and PILP-3, molecular modeling suggested that PILP-2 and PILP-3 shared similar folded structures. Unlike PILP-2, PILP-3 showed a notable activity in abolishing migration and invasion of human neuroblastoma SK-N-SH cells. The ability of PILP-3 to inhibit matrix metalloprotease-2 (MMP-2) activity was higher than that of PILP-2. Pull-down assay revealed protein-protein interaction between PILP-3 and MMP-2. In contrast to mutation on N-terminal region, replacement of amino acids at C-terminus attenuated notably the ability of PILP-3 to inhibit cell invasion, cell migration and MMP-2 activity as well as the binding capability of PILP-3 with MMP-2. Molecular docking showed that N-terminal region of PILP-2 and PILP-3 fitted into the cleft around the active site of MMP-2 catalytic domain. In contrast to that of PILP-2, C-terminal region of PILP-3 was suggested to be in close contact with catalytic domain of MMP-2. Collectively, our data indicate that PILP-3 is a MMP-2 inhibitor and shows an activity in inhibiting migration and invasion of neuroblastoma, and suggest that intact C-terminus is crucial to the activities of PILP-3.

Membrane-damaging activity with A chain and B chain of beta-bungarotoxin.

Beta-bungarotoxin (beta-Bgt) consists of A chain (a phospholipase A2 subunit) and B chain, cross-linked by an intersubunit disulfide bridge. In contrast to a marginal activity noted with beta-Bgt, recombinant A1 chain and B1 chain markedly induced release of calcein from phospholipid vesicles. Reduction of intersubunit disulfide bond by dithiothreitol or glutathione enhanced membrane-damaging activity of beta-Bgt. Moreover, phospholipid-binding capability of recombinant A1 and B1 chains was higher than that of beta-Bgt. In contrast to beta-Bgt, A1 and B1 chains preferably bound lipids with a preference for anionic over zwitterionic phospholipids. Removal of positively charged residues lying on the interface between A chain and B chain resulted in abolishment of membrane-permeabilizing activity of B chain. Taken together, our data indicate that both A and B chains possess the capability to induce vesicle leakage, and reduction of interchain disulfide bond markedly releases this ability from intact beta-Bgt molecule.

Distinctive epidemiologic and clinical features of common krait (Bungarus caeruleus) bites in Sri Lanka.

A prospective study was designed to define epidemiologic and clinical features of krait bites to improve diagnosis, management, and prevention. Among 762 cases of venomous snake bites admitted to 10 Sri Lankan hospitals in which the snake responsible was brought and identified, 88 (11.5%) were caused by common kraits (Bungarus caeruleus). Bites were: most frequent in September through November. Distinctive features of B. caeruleus bites (compared with bites by other species in parentheses) were bitten while sleeping on the ground, 100% (1%); indoors, 100% (49%); between 2300 and 0500 hours, 100% (3%). Only 13% of krait victims were bitten on their lower limbs (82%), only 9% had local swelling (in all cases mild) at the site of the bite (93%), 64% developed respiratory paralysis (2%), and 91% experienced (often severe) abdominal pain (10%). Case fatality was 6% (3%). This distinctive pattern of epidemiology and symptoms will aid clinical recognition (syndromic diagnosis) and prevention of krait bite envenoming.

The organization of the genes encoding the A chains of beta-bungarotoxins: evidence for the skipping of exon.

Bungarus multicinctus (Taiwan banded krait) beta-bungarotoxins consist of two dissimilar polypeptide chains, A and B. The A chain is structurally homologous to phospholipase A(2) (PLA(2)) enzymes. The structural organization of the genes encoding A1, A2 and A8 chains are reported in this study. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with their cDNAs revealed that A1 chain gene organized with four exons and three introns, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponding to the first exon of A1 chain gene is skipped instead of the inclusion of intronic sequence adjacent to the second exon. The resulting A2 chain mRNA encoded a 25 residue signal peptide, which is different from A1 chain mRNA with a 27 residue signal peptide. Nevertheless, expression of the A chain genes was partly regulated by a common mechanism as evidenced by sequence conservation of their promoter region and consensus transcriptional factor binding-sites inside this region. 5'-RACE analyses revealed that A chain mRNAs with 27 residue signal peptide represented the predominant species in the preparation of B. multicinctus venom gland mRNAs. Comparative analyses on PLA(2) genes and cDNAs suggest that this is the first report on the skipping of exon which changes the signal peptide sequence of snake venom proteins.

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus.

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207-232) from Elapidae snake venom glands.

Decreased parasympathetic activities in Malayan krait (Bungarus candidus) envenoming.

Three patients were bitten by the Malayan krait (Bungarus candidus). The patients developed ptosis and generalized muscle weakness which later progressed to respiratory paralysis. All patients showed evidence of decreased parasympathetic activity manifested by mydriasis, hypertension and tachycardia. No specific antivenom was available. All patients received assisted ventilation and supportive treatment. The other forms of treatment included administration of neostigmine, the banded krait (Bungarus fasciatus) antivenom (Thai Red Cross) and plasmapheresis without beneficial response. Two patients recovered. The other patient had permanent brain damage due to anoxia from two episodes of cardiac arrest. While hypertension resolved 6-60 days after admission, mydriasis and tachycardia persisted after discharge in all patients for between 7 days and 2 years. One patient had constipation and defect in micturition which still persisted 2 years after the bite. Decreased parasympathetic activities in Malayan krait bite are perhaps not uncommon and should be examined.

Cloning of a cDNA encoding a nerve growth factor precursor from the Agkistrodon halys Pallas.

Based on the high conservation in the 5' and 3' untranslated regions of NGF cDNAs, oligonucleotides complementary to all these known sequences were synthesized. By RT-PCR, we successfully isolated the complementary DNA encoding NGF precursor from the Agkistrodon halys Pallas (a Chinese snake strain). The nucleotide sequence which presents 90.5%, 88.6% and 63.4% homology to that of Krait Bungarus multicinctus, cobra and human NGF respectively, encoded a prepro-NGF molecule with 241 amino acids and a mature NGF molecule with 119 amino acids. The NGF cDNA inserts were subcloned into pCDNA3 expression vector and then transfected into COS-7 cells. The supernatant of the transfected cells turned out NGF biological activity as assayed by the survival rate of PC12 pheochromocytoma cells.

Genus specific neutralization of Bungarus snake venoms by Thai Red Cross banded krait antivenom.

Thai commercial antivenom raised to Bungarus fasciatus venom neutralized the lethal activity of all Thai Bungarus venoms tested in in vitro neutralization experiments. The neutralizing capacities against B. fasciatus and B. candidus venoms were almost the same, but that against B. flaviceps venom was significantly greater. The efficacy of the antivenom was confirmed in in vivo neutralization experiments also. Results of immunochemical analyses supported results of the animal experiments suggesting the presence of genus specific neutralization.