Proteome analysis of Daboia russelii venom, a medically important snake from the Indian sub-continent.

Daboia russelii is a category-I medically important snake throughout the Indian sub-continent contributing to majority of snakebite incidences in this part of the world. As such, extensive studies on its venom composition and search of efficient and appropriate interventions for its treatment become crucial. In this study, the proteome of Daboia russelii venom from Tanore, Rajshahi, Bangladesh was profiled using a combination of chromatographic and mass spectrometric techniques. A total of 37 different proteins belonging to 11 different snake venom protein families were detected. Proteomics analysis revealed the presence of major phospholipase A2 toxins. Daboiatoxin (both A and B subunits), the main lethal PLA2 toxin in the venom of Daboia siamensis (Myanmar viper) which is neurotoxic, myotoxic and cytotoxic was detected. Presence of Daboxin P, which is a major protein in the venom of Indian Daboia russelii with strong anticoagulant activity, was also observed. Inconsistent distribution of such lethal toxins in the venom of same species calls for more investigations of snake venoms from lesser explored regions and formulation of better alternatives to the current antivenom therapy for efficient treatment.

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.

Evaluating Antivenom Efficacy against Echis carinatus Venoms-Screening for In Vitro Alternatives.

In India, polyvalent antivenom is the mainstay treatment for snakebite envenoming. Due to batch-to-batch variation in antivenom production, manufacturers have to estimate its efficacy at each stage of IgG purification using the median effective dose which involves 100-120 mice for each batch. There is an urgent need to replace the excessive use of animals in snake antivenom production using in vitro alternatives. We tested the efficacy of a single batch of polyvalent antivenom from VINS bioproducts limited on Echis carinatus venom collected from three different locations-Tamil Nadu (ECVTN), Goa (ECVGO) and Rajasthan (ECVRAJ)-using different in vitro assays. Firstly, size-exclusion chromatography (SEC-HPLC) was used to quantify antivenom-venom complexes to assess the binding efficiency of the antivenom. Secondly, clotting, proteolytic and PLA2 activity assays were performed to quantify the ability of the antivenom to neutralize venom effects. The use of both binding and functional assays allowed us to measure the efficacy of the antivenom, as they represent multiple impacts of snake envenomation. The response from the assays was recorded for different antivenom-venom ratios and the dose-response curves were plotted. Based on the parameters that explained the curves, the efficacy scores (ES) of antivenom were computed. The binding assay revealed that ECVTN had more antivenom-venom complexes formed compared to the other venoms. The capacity of antivenom to neutralize proteolytic and PLA2 effects was lowest against ECVRAJ. The mean efficacy score of antivenom against ECVTN was the greatest, which was expected, as ECVTN is mainly used by antivenom manufacturers. These findings pave a way for the development of in vitro alternatives in antivenom efficacy assessment.

Immunorecognition capacity of Indian polyvalent antivenom against venom toxins from two populations of Echis carinatus.

Clinicians report low efficacy of Indian polyvalent antivenom (PAV), with >20 vials required for treatment of a snakebite envenoming. We hypothesize that the antivenom efficacy could be reduced due to insufficient antibodies against some venom toxins. To test this, we used third-generation antivenomics to reveal bound and unbound venom toxins of Echis carinatus venom from Goa (ECVGO) and Tamil Nadu (ECVTN). We used 60, 120, 180, 240, 300, and 360 µg of venom and passed through mini-columns containing ~5 mg Antivenom bound to CNBr beads. The non-retained (unbound) and retained (bound) toxins were identified using reverse-phase HPLC and tandem mass spectrometry. Low molecular weight toxins - Short disintegrins (5.3 kDa) and DIS domain of P-II SVMP from ECVGO and ECVTN showed poor binding with antivenom. The immunorecognition sites of antivenom saturated at the lower antivenom-venom ratio for ECVGO than for ECVTN. The immunoretained capacity of antivenom against ECVTN was 140.6 µg and ECVGO was 125.1 µg. The amount of immunoretained toxins quantified can further be used to estimate the efficacy of antivenom by correlating it with in-vivo studies. The unbound toxins identified from this study could be targeted to improve the effectiveness of antivenom.