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The targeting of specific prey by snake venom toxins is a fascinating aspect of molecular and ecological evolution. Neurotoxic targeting by elapid snakes dominates the literature in this regard; however, recent studies have revealed viper toxins also induce neurotoxic effect. While this effect is thought to primarily be driven by prey selectivity, no study has quantified the taxonomically specific neurotoxicity of the viper clade consisting of Daboia, Macrovipera, Montivipera, and Vipera genera. Here, we tested venom toxin binding from 28 species of vipers from the four genera on the alpha 1 neuronal nicotinic acetylcholine receptors (nAChRs) orthosteric sites of amphibian, avian, lizard, rodent, and human mimotopes (synthetic peptides) using the Octet HTX biolayer interferometry platform. Daboia siamensis and D. russelii had broad binding affinity towards all mimotopes, while D. palestinae had selectivity toward lizard. Macrovipera species, on the other hand, were observed to have a higher affinity for amphibian mimotopes except for M. schweizeri, which inclined more toward lizard mimotopes. All Montivipera and most Vipera species also had higher affinity toward lizard mimotopes. Vipera a. montandoni, V. latastei, V. nikolski, and V. transcaucasina had the least binding to any of the mimotopes of the study. While a wide range of affinity binding towards various mimotopes were observed within the clade, the lowest affinity occurred towards the human target. Daboia siamensis and Macrovipera lebetina exhibited the greatest affinity toward the human mimotope, albeit still the least targeted of the mimotopes within those species. Overlaying this toxin-targeting trait over phylogeny of this clade revealed multiple cases of amplification of this trait and several cases of secondary loss. Overall, our results reveal dynamic variation, amplification, and some secondary loss of the prey targeting trait by alpha-neurotoxins within the venoms of this clade, indicating evolutionary selection pressure shaping the basic biochemistry of these venoms. Our work illustrates the successful use of this biophysical assay to further research snake venom neurotoxins and emphasizes the risk of generalizing venom effects observed on laboratory animals to have similar effects on humans.

Critically ill COVID-19 patients present an inflammatory and procoagulant status with a high rate of relevant macro- and microvascular thrombosis. Furthermore, high rates of heparin resistance have been described; yet, individualized anticoagulation by drug monitoring has not been sufficiently researched. We analyzed data from critically ill COVID-19 patients treated at Innsbruck Medical University Hospital with routinely adapted low-molecular-weight heparin (LMWH) doses according to anti-Xa peak levels, and regularly performed ClotPro analyses (a viscoelastic hemostatic whole blood test). A total of 509 anti-Xa peak measurements in 91 patients were categorized as below ( 0.012 IU/mL/mg) expected ranges with respect to the administered LMWH doses. Besides intergroup comparisons, correlations between anti-Xa levels and ClotPro clotting times (CTs) were performed (226 time points in 84 patients). Anti-Xa peak levels remained below the expected range in the majority of performed measurements (63.7%). Corresponding patients presented with higher C-reactive protein and D-dimer but lower antithrombin levels when compared with patients achieving or exceeding the expected range. Consequently, higher enoxaparin doses were applied in the sub-expected anti-Xa range group. Importantly, 47 (51.6%) patients switched between groups during their intensive care unit (ICU) stay. Anti-Xa levels correlated weakly with IN test CT and moderately with Russell's viper venom (RVV) test CT. Critically ill COVID-19 patients present with a high rate of LMWH resistance but with a variable LMWH response during their ICU stay. Therefore, LMWH–anti-Xa monitoring seems inevitable to achieve adequate target ranges. Furthermore, we propose the use of ClotPro's RVV test to assess the coagulation status during LMWH administration, as it correlates well with anti-Xa levels but more holistically reflects the coagulation cascade than anti-Xa activity alone.

Snakebite envenomation is a high priority neglected tropical disease that predominantly affects rural communities living in developing countries. Due to myriad of complications including coagulopathies, neurotoxicity, nephrotoxicity and local tissue destruction, treating snakebite victims is a major challenge for clinicians. Russell’s viper (Daboia russelii) is one of the ‘Big Four’ venomous snakes in India, and it is responsible for the most snakebite-induced deaths and disabilities. Acute kidney injury occurs frequently following Russell’s viper bites and it is a critical factor contributing to disabilities, deaths and excessive treatment costs. In addition to commonly observed envenomation effects, Russell’s viper bites induce some rare complications such as priapism, sialolithiasis and splenic rupture. Here, we report a case of Wunderlich syndrome that developed in a 22-year-old male following a Russell’s viper bite. The patient displayed severe coagulopathies, abdominal tenderness, and hypotension. Notably, a peri-nephric haematoma was identified through ultrasound and computerised tomographic imaging. The haemorrhage was successfully treated using angioembolisation, and the patient recovered without any difficulties. Although a clinical condition such as this is rare, it is important to create awareness among treating clinicians about its occurrence, diagnosis and clinical management.

Envenomation by Cerastes cerastes often results in local dermonecrotic lesions. While immunotherapy is effective in reversing systemic symptoms, this strategy remains deficient in counteracting the extended dermonecrosis induced from the bite site. In this study, the therapeutic effect of pharmacological drugs on the dermonecrotic activity of the venom was investigated. Venom administration caused a marked dermonecrotic lesion with increased levels of oxidative stress biomarkers (MPO, EPO, NO, H

The most common venomous snake in Israel, both in geographic spread and in number of snakebite incidents, is Daboia (Vipera) palaestinae. The clinical presentation of D. palaestinae envenomation varies and includes both local and systemic symptoms. Studies conducted on D. palaestinae revealed different amounts of venom in the snakes' glands in different seasons, however little is known regarding the potential impact of this finding on the clinical presentation after D. palaestinae bites during different seasons.To evaluate whether there is a difference in the severity of the clinical presentation of D. palaetinae bites in different seasons.A retrospective chart review study including all patients diagnosed with D. palaestinae bites treated at Shamir Medical Center from 2006 through 2020. Patients were divided into two groups: early bite season - spring and early summer, and late bite season - late summer and autumn. Variables examined included demographic features, admission details and treatment administrated.One hundred and seven D. palaestinae bite victims were included, forty-five were bitten during the early season and sixty-two during the late season. Four patients in the early season (8.9%) and one patient (1.6%) in the late season presented with decreased level of consciousness, and four patients, all from the early season group, required mechanical ventilation (p 0.05) Vasopressors were used in six patients (13.3%) during the early season and two (3.2%) during the late season; (p = 0.06). There were no other differences between the groups, except for a lowest platelet count during hospitalization (mean 161.5 ± 51 K/μl during early season and 196.9 ± 77 K/μl during late season (p 0.01).D. palaestinae bite victims more often present as critically ill patients during the spring and early summer compared to late summer and autumn. Hospitals should be prepared with appropriate staff training and medications for treating such patients, especially during the early season. However, in general, D. palaestinae bites are as dangerous during the late season as they are during the early season, and all snake bite victims should be treated with a high index of suspicion regardless of the season.

Splenic rupture and/or splenectomy is/are not uncommon in clinical arena. Here we present this case of extensive haemorrhage-induced splenic rupture which resulted in splenectomy in a young healthy male (who did not have any previous medical conditions) following a Russell's viper bite. He developed upper abdominal and shoulder pain on his left side along with hypotension and reduced level of haemoglobin on the third day following bite despite antivenom treatment. Following confirmation of splenic rupture and haemoperitoneum by ultrasound and computed tomography scans, an emergency splenectomy was performed using laparotomy. Although Russell's viper bites are known to induce bleeding complications, splenic rupture due to haemorrhage in spleen has not been previously reported. Russell's viper venom toxins such as metalloproteases, serine proteases and phospholipase A2 might have affected the vascular permeability resulting in excessive bleeding and increased pressure in the spleen leading to rupture. Further investigations are required to underpin the impact of snake venom toxins on the architecture and functions of spleen. However, the clinicians who treat snakebites should be aware of this type of rare complications so as to provide appropriate management for such victims.

The Trimeresurus complex consists of diverse medically important venomous pit vipers that cause snakebite envenomation. Antivenoms, however, are in limited supply, and are specific to only two out of the many species across Asia. This study thus investigated the immunoreactivities of regional pit viper antivenoms toward selected Trimeresurus pit viper venoms, and examined the neutralization of their hemotoxic activities. Trimeresurus albolabris Monovalent Antivenom (TaMAV, Thailand) exhibited a higher immunoreactivity than Hemato Bivalent Antivenom (HBAV, raised against Trimeresurus stejnegeri and Protobothrops mucrosquamatus, Taiwan) and Gloydius brevicaudus Monovalent Antivenom (GbMAV, China), attributed to its monovalent nature and conserved antigens in the Trimeresurus pit viper venoms. The venoms showed moderate-to-strong in vitro procoagulant and in vivo hemorrhagic effects consistent with hemotoxic envenomation, except for the Sri Lankan Trimeresurus trigonocephalus venom which lacked hemorrhagic activity. TaMAV was able to differentially neutralize both in vitro and in vivo hemotoxic effects of the venoms, with the lowest efficacy shown against the procoagulant effect of T. trigonocephalus venom. The findings suggest that TaMAV is a potentially useful treatment for envenomation caused by hetero-specific Trimeresurus pit vipers, in particular those in Southeast Asia and East Asia. Clinical study is warranted to establish its spectrum of para-specific effectiveness, and dosages need be tailored to the different species in respective regions.

Inefficacy and associated risks of current antivenom has raised the need for alternative approaches of snakebite management. Aptamers are one such alternative which is being pursued for therapeutic interventions as well as for design of diagnostic kits due to its high specificity. Present study focussed on designing and validating nucleic acid aptamers against snake venom PLA2, a hydrolytic enzyme present in all venomous snakes. The aptamers were designed by adding nucleic acid chain on the surface of Daboxin P, a major PLA2 enzyme of Daboia russelii venom. Binding characteristics of the aptamers were confirmed by docking to Daboxin P as well as acidic and basic PLA2s from different snake species using in silico docking. The aptamers folded into different tertiary structures and bound to the active and Ca2+ binding site of PLA2 enzymes. Molecular dynamics simulation analysis of Daboxin P-aptamer complexes showed that the complexes were stable in an aqueous environment. The electrophoretic mobility shift assay further confirmed the binding of the synthetic aptamers to Daboxin P and other snake venom PLA2 enzymes. The aptamers inhibited the sPLA2 activity with an IC50 value ranging between 0.52 μM and 0.77 μM as well as the anticoagulant activity of Daboxin P. The aptamers could also inhibit the PLA2 activity of Echis carinatus crude venom and anti-coagulant activity of Bungarus caeruleus crude venom, members of big four snakes. However, the aptamers didn't inhibit fibrinogenolytic or proteolytic activity of big four venom as well as the coagulation and hemolytic activities. Thus, aptamers can be rationally designed to inhibit the biochemical and biological activities of snake venom proteins.

Lebetin 2 (L2), a natriuretic-like peptide (NP), exerts potent cardioprotection in myocardial infarction (MI), with stronger effects than B-type natriuretic peptide (BNP). To determine the molecular mechanisms underlying its cardioprotection effect, we used molecular modeling, molecular docking and molecular dynamics (MD) simulation to describe the binding mode, key interaction residues as well as mechanistic insights into L2 interaction with NP receptors (NPRs). L2 binding affinity was determined for human, rat, mouse and chicken NPRs, and the stability of receptor–ligand complexes ascertained during 100 ns-long MD simulations. We found that L2 exhibited higher affinity for all human NPRs compared to BNP, with a rank preference for NPR-A > NPR-C > NPR-B. Moreover, L2 affinity for human NPR-A and NPR-C was higher in other species. Both docking and MD studies revealed that the NPR-C–L2 interaction was stronger in all species compared to BNP. Due to its higher affinity to human receptors, L2 could be used as a therapeutic approach in MI patients. Moreover, the stronger interaction of L2 with NPR-C could highlight a new L2 signaling pathway that would explain its additional effects during cardiac ischemia. Thus, L2 is a promising candidate for drug design toward novel compounds with high potency, affinity and stability.

The African viperid snake genera

The COVID-19 pandemic caused by SARS-CoV-2 requires new treatments both to alleviate the symptoms and to prevent the spread of this disease. Previous studies demonstrated good antiviral and virucidal activity of phospholipase A2s (PLA2s) from snake venoms against viruses from different families but there was no data for coronaviruses. Here we show that PLA2s from snake venoms protect Vero E6 cells against SARS-CoV-2 cytopathic effects. PLA2s showed low cytotoxicity to Vero E6 cells with some activity at micromolar concentrations, but strong antiviral activity at nanomolar concentrations. Dimeric PLA2 from the viper Vipera nikolskii and its subunits manifested especially potent virucidal effects, which were related to their phospholipolytic activity, and inhibited cell–cell fusion mediated by the SARS-CoV-2 spike glycoprotein. Moreover, PLA2s interfered with binding both of an antibody against ACE2 and of the receptor-binding domain of the glycoprotein S to 293T/ACE2 cells. This is the first demonstration of a detrimental effect of PLA2s on β-coronaviruses. Thus, snake PLA2s are promising for the development of antiviral drugs that target the viral envelope, and could also prove to be useful tools to study the interaction of viruses with host cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03985-6.

Venomous snakebites cause clinical manifestations that range from local to systemic and are considered a significant global health challenge. Persistent or refractory thrombocytopenia has been frequently reported in snakebite patients, especially in cases caused by viperidae snakes. Viper envenomation-induced thrombocytopenia may persist in the absence of significant consumption coagulopathy even after the antivenom treatment, yet the mechanism remains largely unknown. Our study aims to investigate the mechanism and discover novel therapeutic targets for coagulopathy-independent thrombocytopenia caused by viper envenomation. Here we found that patients bitten by Protobothrops mucrosquamatus and Trimeresurus stejnegeri, rather than Naja. atra may develop antivenom-resistant and coagulopathy-independent thrombocytopenia. Crude venoms and the derived C-type lectin-like proteins from these vipers significantly increased platelet surface expression of neuraminidase and platelet desialylation, therefore led to platelet ingestion by both macrophages and hepatocytes in vitro, and drastically decreased peripheral platelet counts in vivo. Our study is the first to demonstrate that desialylation-mediated platelet clearance is a novel mechanism of viper envenomation-induced refractory thrombocytopenia and C-type lectin-like proteins derived from the viper venoms contribute to snake venom-induced thrombocytopenia. The results of this study suggest the inhibition of platelet desialylation as a novel therapeutic strategy against viper venom-induced refractory thrombocytopenia.

Venom pathology is not restricted to the direct toxic effects of venom. Immunoinflammatory alteration as the etiology of snake venom-induced acute kidney injury (SAKI) is a less trodden path toward the development of alternative therapeutic approach. In the present study, we have associated the crest of renal damage stage to the immunological alteration, as reflected in thymic and peripheral T cell polarization in the murine model of SAKI. Renal injury in mice was confirmed from significant dysuresis and adversely altered biochemical renal markers. Histopathological alterations, as revealed by marked tubular and glomerular damage, reaffirmed kidney injury. SAKI is accompanied by significant inflammatory changes as indicated by neutrophilic leucocytosis, increased neutrophil to lymphocyte ratio and plasma CRP levels. Thymic immunophenotyping revealed significantly increased CD8

Abdominal pain is known to be an early clinical predictor of severe systemic Russell's viper (RV) envenomation and is often associated with the later development of coagulopathy and neurotoxicity. The mechanism of abdominal pain is unknown, but we postulated it might be due to intestinal microvascular endothelial gut damage. Gut-toxicity can be detected using the novel biomarker Intestinal Fatty Acid Binding Protein (IFABP). We also wanted to explore the mechanisms and consequences of this toxicity by measuring procalcitonin as a specific marker of sepsis triggered by bacterial endotoxin, and serum cystatin-C (CysC) as a measure of acute kidney injury. We hypothesised that severe gut-injury might lead to gut-barrier failure, translocation of gastrointestinal microorganisms, associated sepsis and systemic inflammatory response syndrome (SIRS), with a possible exacerbation of snake-bite severity, including acute kidney injury that was previously attributed to direct venom effects.Serial plasma samples previously collected from 16 RV envenomations with abdominal pain, 15 RV envenomations without abdominal pain and 25 healthy controls were assayed for IFABP. A subgroup of these RV envenomations were assayed for procalcitonin (The median peak IFABP for RV envenomations was much higher than healthy controls [3703.0 pg/mL (IQR 2250.1-13702.0 pg/mL) vs. 270.1 pg/mL (IQR 153.5-558.0 pg/mL) (IFABP is significantly elevated indicating enterocyte damage occurs in RV envenomation. IFABP correlated with markers of sepsis (procalcitonin) and acute kidney injury (serum CysC) suggesting that enterocyte damage resulting in translocation of microbial associated molecular patterns (MAMPs) contributes to RV envenomation associated SIRS and sepsis.

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by

Antivenom is an essential treatment for snake envenomation; however, early adverse reactions (EARs) are major limitations to its use. We performed a retrospective cross-sectional study using Ramathibodi Poison Center data (January 2016 to December 2017) to clarify the incidence and severity of EARs following different F(ab’)2 antivenoms. Among 1006 envenomed patients, 684 (68%) received antivenom therapy with a total of 1157 doses, mostly green pit viper antivenom. The overall EAR incidence and rate were 22. 5% (154/684) and 15% (173/1157), respectively. The EAR rate following each type of antivenom was >10%, except for Russell’s viper antivenom (2.9%); the severe reaction rate was 2.6% (30/1157). Malayan pit viper bites caused a high incidence of EARs (37.8%) and the highest EAR rate (22.3%). Fifty-two cases developed anaphylaxis. All EARs occurred within 2 h after treatment initiation. No deaths were attributed to EARs. The duration of administration was significantly different between doses of antivenom that induced EARs and those that did not. In conclusion, all types and every dose of antivenom should be infused for 30–60 min. Preparation of resuscitation equipment and continuous clinical observation are crucial for at least 2 h after administration, and prompt treatment should be provided when EARs occur.

Among the medically most important snakes in the world, the species belonging to the genus

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom's toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude

Background Venomous snake bites cause acute medical emergencies and are fatal. India accounts for large proportion of snake-bite deaths globally. Medically important ‘BIG FOUR’ snakes of India are Bungarus caeruleus (krait), Naja naja (cobra), Echis carinatus (saw-scaled viper) and Daboia russelii (Russell's viper). Polyherbal formulations have been proved to be effective in treatment of diseases than a single formulation. Objective(s) To evaluate aqueous ethanolic extract cocktail of Azadirachata indica, Butea monosperma, Citrus limon, Clerodendrum serratum and Areca catechu for antidote potential against BIG FOUR venoms in ex vivo and in vivo model. Materials and methods Anti-hemorrhagic and venom neutralization studies were performed in seven-day old chick embryo model for ex vivo studies. In vivo studies were performed using male Swiss albino mice for antivenom potential of herbal cocktail by performing anti-edematic, anti-hemorrhagic, anti-myotoxic activity, and venom neutralization. Results Herbal cocktail exhibited differential venom inhibition potential against four venoms tested. Hemorrhagic activity was completely neutralized by the herbal cocktail; myotoxic activities of krait and Russell's viper venom were neutralized; while anti-edematic activity was observed for krait and cobra venom. Herbal cocktail completely neutralized venom lethality (3∗LD50) of krait and saw-scaled viper venom. Conclusion Inhibitions of various venom components of all four venoms suggests presence of phytochemicals in herbal cocktail with therapeutic properties. Further studies would help in the development of a formulation as a first-aid towards treatment of snake bite victims.

In this study, we examined the potential use of N-acetyl-L-cysteine (NAC) in association with a polyvalent antivenom and as stand-alone therapy to reduce the acute local and systemic effects induced by Lachesis muta muta venom in rats. Male Wistar rats (300–350 g) were exposed to L. m. muta venom (1.5 mg/kg – i.m.) and subsequently treated with anti-Bothrops/Lachesis serum (antivenom:venom ratio 1:3 ‘v/w’ – i.p.) and NAC (150 mg/kg – i.p.) separately or in association; the animals were monitored for 120 min to assess changes in temperature, locomotor activity, local oedema formation and the prevalence of haemorrhaging. After this time, animals were anesthetized in order to collect blood samples through intracardiac puncture and then euthanized for collecting tissue samples; the hematological-biochemical and histopathological analyses were performed through conventional methods. L. m. muta venom produced pronounced local oedema, subcutaneous haemorrhage and myonecrosis, with both antivenom and NAC successfully reducing the extent of the myonecrotic lesion when individually administered; their association also prevented the occurrence of subcutaneous haemorrhage. Venom-induced creatine kinase (CK) release was significantly prevented by NAC alone or in combination with antivenom; NAC alone failed to reduce the release of hepatotoxic (alanine aminotransferase) and nephrotoxic (creatinine) serum biomarkers induced by L. m. muta venom. Venom induced significant increase of leucocytes which was also associated with an increase of neutrophils, eosinophils and monocytes; antivenom and NAC partially reduced these alterations, with NAC alone significantly preventing the increase of eosinophils whereas neither NAC or antivenom prevented the increase in monocytes. Venom did not induce changes in the erythrogram parameters. In the absence of a suitable antivenom, NAC has the potential to reduce a number of local and systemic effects caused by L. m. muta venom.

Background: The snake venom nerve growth factor (NGF)-induced signal transduction mechanism has never been explored.Research design and methods: Homology modeling and molecular dynamic studies of the interaction between Russell's viper venom NGF (RVV-NGFa) and mammalian tropomyosin-receptor kinase A (TrkA) was done by computational analysis. Transcriptomic and quantitative tandem mass spectrometry analyses determined the expression of intracellular genes and proteins, respectively, in RVV-NGFa-treated PC-12 neuronal cells. Small synthetic inhibitors of the signal transduction pathways were used to validate the major signaling cascades of neuritogenesis by RVV-NGFa.Results: A comparative computational analysis predicted the binding of RVV-NGFa, mouse 2.5S-NGF (conventional neurotrophin), and Nn-α-elapitoxin-1 (non-conventional neurotrophin) to different domains of the TrkA receptor in PC-12 cells. The transcriptomic and quantitative proteomic analyses in unison showed differential expressions of common and unique genes and intracellular proteins, respectively, in RVV-NGFa-treated cells compared to control (untreated) mouse 2.5S-NGF and Nn-α-elapitoxin-1-treated PC-12 cells. The RVV-NGFa primarily triggered the mitogen-activated protein kinase-1 (MAPK1) signaling pathway for inducing neuritogenesis; however, 36 pathways of neuritogenesis were uniquely expressed in RVV-NGFa-treated PC-12 cells compared to mouse 2.5S NGF or Nn-α-elapitoxin-1 treated cells.Conclusion: The common and unique intracellular signaling pathways of neuritogenesis by classical and non-classical neurotrophins were identified.

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell’s viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell’s viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.