Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

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.

Molecular phylogeny reveals distinct evolutionary lineages of the banded krait, Bungarus fasciatus (Squamata, Elapidae) in Asia.

The banded krait, Bungarus fasciatus is a widespread elapid snake, likely to comprise several distinct species in different geographic regions of Asia. Therefore, based on molecular phylogenetics and comparative morphology data, we present an overview of the systematic composition of the species to delimit potential biogeographic boundaries. Our phylogenetic analyses, based on four mitochondrial genes, reveal the existence of at least three evolutionary lineages within B. fasciatus, corresponding to Indo-Myanmar, Sundaic and eastern Asian lineages. We are convinced that there are at least three taxonomic entities within the nomen B. fasciatus and restrict the distribution of B. fasciatus sensu stricto to the Indo-Myanmar region. We also provide additional natural history data of the taxon from eastern India. Finally, we advocate further studies to establish the degree of reproductive isolation among these diverging evolutionary lineages and to reassess the systematic status of this species complex especially the Sundaic and eastern Asian lineages.

Proteome Decomplexation of Trimeresurus erythrurus Venom from Mizoram, India.

Green pit vipers are the largest group of venomous vipers in tropical and subtropical Asia, which are responsible for most of the bite cases across this region. Among the green pit vipers of the Indian subcontinent, Trimeresurus erythrurus is the most prevalent; however, limited knowledge is available about its venomics. Proteome decomplexation of T. erythrurus venom using mass spectrometry revealed a blend of 53 different proteins/peptides belonging to 10 snake venom protein families. Phospholipase A2 and snake venom serine proteases were found to be the major enzymatic families, and Snaclec was the major nonenzymatic family in this venom. These protein families might be responsible for consumptive coagulopathy in victims. Along with these, snake venom metalloproteases, l-amino acid oxidases, disintegrins, and cysteine-rich secretory proteins were also found, which might be responsible for inducing painful edema, tissue necrosis, blistering, and defibrination in patients. Protein belonging to C-type lectins, C-type natriuretic peptides, and glutaminyl-peptide cyclotransfreases were also observed as trace proteins. The crude venom shows platelet aggregation in the absence of any agonist, suggesting their role in alterations in platelet functions. This study is the first proteomic analysis of T. erythrurus venom, contributing an overview of different snake venom proteins/peptides responsible for various pathophysiological disorders obtained in patients. Data are available via ProteomeXchange with the identifier PXD038311.

Venom of several Indian green pit vipers: Comparison of biochemical activities and cross-reactivity with antivenoms.

Green pit vipers, a name that can refer to several unrelated species, comprise a large group of venomous snakes found across the humid areas of tropical and sub-tropical Asia, and are responsible for most of the bite cases across this region. In India, green pit vipers belonging to several genera are prevalent in the northern and north-eastern hilly region, unrelated to species present in the peninsular region. In the present study, crude venom of representative species of green pit vipers present in the north and north-eastern hilly region of India (Trimeresurus erythrurus, T. septentrionalis, Viridovipera medoensis, and Popiea popieorum) were characterized to elucidate venom composition and venom variation. Profiling of crude venoms using SDS-PAGE and RP-HPLC methods revealed quantitative differences among the species. Further, in vitro biochemical assays reveal variable levels of phospholipase activity, coagulation activity, thrombin-like activity, fibrinogenolytic and haemolytic activity. This correlates with the pseudo-procoagulant effects on the haemostatic system of victims, which causes consumptive coagulopathy, frequently observed in patients bitten by green pit vipers. The immunoreactivity of Indian polyvalent antivenom and Thai green pit viper antivenom towards crude venoms were also evaluated by western blotting and inhibition of biochemical activities. The results exhibited poor efficacy of Indian polyvalent antivenom in neutralizing the venom toxins of crude venoms; however, Thai green pit viper antivenin (raised against the venom of Trimeresurus allbolabris, not present in India) showed higher immunoreactivity towards congeneric venoms tested. Analysis of green pit viper bite patients records from a community health centre in Assam, India, further revealed the inability of Indian polyvalent antivenom to reverse the extended coagulopathy featured.

A paper microfluidic device based colorimetric sensor for the detection and discrimination of elapid versus viper envenomation.

Snake bites are a neglected tropical disease, causing mortality and severe damage to various vital organs like the nervous system, kidneys and heart. There is increasing interest in designing new antivenom treatments that are more specific to particular groups (either taxonomic or regional) of species, given the increasing evidence that current polyvalent Indian antivenom is ineffective in many situations. Under these circumstances, being able to detect the species, or a group of species, responsible for the envenomation becomes important. Unfortunately, no such diagnostic tool is available in the Indian market. Such a tool will need to be rapid, sensitive and affordable. To address this need, we have combined the power of nanotechnology and paper microfluidics and herein report a device that has the ability to detect and differentiate viper venom from elapid and scorpion venom. In principle, this assay is based on the release of the dye from the stimuli-responsive glutaraldehyde cross-linked methylene blue-loaded gelatin (GMG) nanoparticles in the presence of snake venom metalloproteases and serine proteases. The developed equipment-free assay can detect and discriminate viper venom from that of elapids and scorpions. The low-end detection limit of the sensor is ∼3.0 ng for the saw-scaled viper Echis carinatus, while the same for Russell's viper Daboia russelii is ∼6.0 ng. The performance of the sensor remains unaltered for different batches of GMG nanoparticles. Altogether, this finding establishes the role of nanotechnology and paper microfluidics in the rapid and accurate detection of viper venom.

Molecular phylogenetic analysis of the genus Gloydius (Squamata, Viperidae, Crotalinae), with description of two new alpine species from Qinghai-Tibet Plateau, China.

We provide a molecular phylogeny of Asian pit vipers (the genus Gloydius) based on four mitochondrial genes (12S, 16S, ND4, and cytb). Sequences of Gloydiushimalayanus, the only member of the genus that occurs south of the Himalayan range, are included for the first time. In addition, two new species of the genus Gloydius are described based on specimens collected from Zayu, Tibet, west of the Nujiang River and Heishui, Sichuan, east of the Qinghai-Tibet Plateau. The new species, Gloydiuslipipengi sp. nov., can be differentiated from its congeners by the combination of the following characters: the third supralabial not reaching the orbit (separated from it by a suborbital scale); wide, black-bordered greyish postorbital stripe extending from the posterior margin of the orbit (not separated by the postoculars, covering most of the anterior temporal scale) to the ventral surface of the neck; irregular black annular crossbands on the mid-body; 23-21-15 dorsal scales; 165 ventral scales, and 46 subcaudal scales. Gloydiusswild sp. nov. can be differentiated from its congeners by the narrower postorbital stripe (only half the width of the anterior temporal scale, the lower edge is approximately straight and bordered with white); a pair of arched stripes on the occiput; lateral body lakes black spots; a pair of round spots on the parietal scales; 21 rows of mid-body dorsal scales; zigzag dark brown stripes on the dorsum; 168-170 ventral scales, and 43-46 subcaudal scales. The molecular phylogeny in this study supports the sister relationship between G.lipipengi sp. nov. and G.rubromaculatus, another recently described species from the Qinghai-Tibet Plateau, more than 500 km away, and indicate the basal position of G.himalayanus within the genus and relatively distant relationship to its congeners.

Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base.

Snakebite incidence at least partly depends on the biology of the snakes involved. However, studies of snake biology have been largely neglected in favour of anthropic factors, with the exception of taxonomy, which has been recognised for some decades to affect the design of antivenoms. Despite this, within-species venom variation and the unpredictability of the correlation with antivenom cross-reactivity has continued to be problematic. Meanwhile, other aspects of snake biology, including behaviour, spatial ecology and activity patterns, distribution, and population demography, which can contribute to snakebite mitigation and prevention, remain underfunded and understudied. Here, we review the literature relevant to these aspects of snakebite and illustrate how demographic, spatial, and behavioural studies can improve our understanding of why snakebites occur and provide evidence for prevention strategies. We identify the large gaps that remain to be filled and urge that, in the future, data and relevant metadata be shared openly via public data repositories so that studies can be properly replicated and data used in future meta-analyses.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

Rational truncation of aptamer for cross-species application to detect krait envenomation.

In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent).