Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A2, L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.

The lethal and enzymatic activities of venom from Naja sumatrana (Equatorial spitting cobra) were determined and compared to venoms from three other Southeast Asian cobras (Naja sputatrix, Naja siamensis and Naja kaouthia). All four venoms exhibited the common characteristic enzymatic activities of Asiatic cobra venoms: low protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase activities, moderately high acetylcholinesterase and hyaluronidase activities and high phospholipase A2. Fractionation of N. sumatrana venom by Resource® S cation exchange chromatography (GE Healthcare, USA) yielded nine major protein peaks, with all except the acidic protein peak being lethal to mice. Most of the protein peaks exhibit enzymatic activities, and L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, 5'-nucleotidase and hyaluronidase exist in multiple forms. Comparison of the Resource® S chromatograms of the four cobra venoms clearly indicates that the protein composition of N. sumatrana venom is distinct from venoms of the other two spitting cobras, N. sputatrix (Javan spitting cobra) and N. siamensis (Indochinese spitting cobra). The results support the revised systematics of the Asiatic cobra based on multivariate analysis of morphological characters. The three spitting cobra venoms exhibit two common features: the presence of basic, potentially pharmacologically active phospholipases A2 and a high content of polypeptide cardiotoxin, suggesting that the pathophysiological actions of the three spitting cobra venoms may be similar.

Bungarus flaviceps (red-headed krait) venom presents an intravenous LD50 of 0.32 μg/g and exhibits enzymatic activities similar to other Bungarus toxins. ELISA cross-reactions between anti-Bungarus flaviceps and a variety of elapid and viperid venoms were observed in the current study. Double-sandwich ELISA was highly specific, since anti-B. flaviceps serum did not cross-react with any tested venom, indicating that this assay can be used for species diagnosis in B. flaviceps bites. In the indirect ELISA, anti-B. flaviceps serum cross-reacted moderately with three different Bungarus venoms (9-18%) and Notechis scutatus venom, but minimally with other elapid and viperid toxins. The results indicated that B. flaviceps venom shares common epitopes with other Bungarus species as well as with N. scutatus. The lethality of the B. flaviceps venom was neutralized effectively by antiserum prepared against B. candidus and B. flaviceps toxins and a commercial bivalent elapid antivenom prepared against B. multicinctus and Naja naja atra venoms, but was not neutralized by commercial antivenoms prepared against Thai cobra, king cobra and banded krait. These data also suggested that the major lethal toxins of B. flaviceps venom are similar to those found in B. multicinctus and B. candidus venoms.

The serum kinetics of Calloselasma rhodostoma (Malayan pit viper) venom - specifically two of its components, the major hemorrhagin (rhodostoxin) and a thrombin-like enzyme - was examined in a rabbit by double-sandwich enzyme-linked immunosorbent assay (ELISA). The animal received intramuscularly a 1.0-mg/kg dose of C. rhodostoma venom. The venom level in serum peaked 12 hours after the injection, followed by a gradual decline and finally reached low rates 72 hours after administration. The serum kinetic profile of venom components, however, did not correspond to the profile of the whole C. rhodostoma venom. The serum levels of the C. rhodostoma thrombin-like enzyme increased slowly and peaked only 48 hours post-injection. Then both thrombin-like enzyme and rhodostoxin remained at relatively high levels 72 hours after administration. Data suggest that various venom components bind to tissue at the injection site with different affinities and that conjugated venom components were continuously released into circulation at different rates. The prolonged high serum levels of both thrombin-like enzyme and hemorrhagin are consistent with the clinical picture of prolonged clotting deficiency in severe cases of C. rhodostoma envenomation. Our results also suggest that since venom components are being released into and eliminated from the circulation at different rates, the "average composition" of the venom antigen in the circulation changes over time. This implies that data from ELISA quantification of antigen levels from serum venom employing "whole venom" as reagent must be interpreted with care.