Venom of several Indian green pit vipers: Comparison of biochemical activities and cross-reactivity with antivenoms
Graphical abstract
Introduction
The Trimeresurus radiation, belonging to the Crotalinae subfamily of the family Viperidae, comprises the largest group of venomous snakes in tropical and sub-tropical Asia. This group consists of around 55 species (listed under Trimeresurus and Craspedocephalus in www.reptile-database) that were reclassified into different genera (Malhotra and Thorpe, 2004), which some authors prefer to treat as subgenera (David et al., 2011). Their distribution ranges from the Indian subcontinent throughout Southeast Asia, southern China and the Indo-Malayan and Philippine archipelagos. A large number of species have a characteristic bamboo green to yellow body colour and are generally described as “Green Pit Vipers”. The literature on green pit viper bites in Southeast Asian countries (including Thailand, China, Sri Lanka, Singapore, Vietnam, Nepal) suggests their medical importance and considerable role in global snakebite epidemiology (Blessmann et al., 2018; Fuchs et al., 2019; Mong and Tan, 2016; Pandey et al., 2019; Rathnayaka et al., 2017; Zeng et al., 2019). Clinical manifestations observed in victims of green pit viper envenomation include local symptoms such as extensive swelling of bite site, mild dyspnoea, nausea and significant pain, followed by systemic toxicity including tissue necrosis and haematological abnormalities (such as consumptive coagulopathy, thrombocytopenia) as well as acute kidney injury (AKI), suggesting the haemotoxic nature of their venoms (Fuchs et al., 2019; Greene et al., 2017; Pandey et al., 2019). Also, aphasia, hemiplegia, speech disturbance, local erythema, ecchymosis and acute cerebral infarction have also been reported following green pit viper bites (Zeng et al., 2019). A few envenomation cases have been reported from Himachal Pradesh, India, and in Solan district, green pit vipers were implicated in 70% of cases where the biting snake was identified, and coagulopathy was reported in about 50% of cases, with swelling in about 70% of cases (Gupt et al., 2015). A review of 30 paediatric snakebite cases in Shimla, Himachal Pradesh (Sood et al., 2020) also reported a high incidence of haemolytic envenomation (87.5%) involving thrombocytopenia (observed in 26.7% of cases), prolonged PT/INR (76.6%) and prolonged activated partial thromboplastin time (36.7%), as well as swelling in 80.9% of cases. Further a case study report by Gharsangi and co-worker in 2019, documented prolonged coagulopathy which was unresponsive to antivenom. The patients potentially envenomated with Himalayan pit viper developed neurological disorder diplopia, which is uncommon in pit viper bites (Gharsangi et al., 2019). Based on these collective clinical manifestations and knowledge of distribution of species, these envenomation cases are most likely to be caused by T. septentrionalis, which is the only green pit viper present in Himachal Pradesh. In less severe cases, symptomatic therapy is the preferred treatment; however, in many cases antivenom is required to treat the patients with green pit viper bites (Greene et al., 2017; Mong and Tan, 2016; Zeng et al., 2019).
Various studies have been conducted to assess the neutralizing potential and cross reactivity of country-specific antivenoms towards various green pit viper venoms. Tan et al. studied the venom toxicity as well as cross-reactivity of Popeia nebularis venom towards Thai Green Pit Viper monovalent antivenom (GPVAV) and reported efficient neutralization (Tan et al., 2019). Previously, they had reported cross-reactivity and efficient neutralization of Indonesian Trimeresurus complex by GPVAV compared to Indonesian trivalent antivenom Biosave® Serum Anti Bisa Ular (Tan et al., 2017), in which the only viper venom used in the immunization mix is the distantly related Malayan pit viper Calloselasma rhodostoma. Similar results were shown with Trimeresurus albolabris, Trimeresurus purpureomaculatus, Craspedocephalus wiroti and Craspedocephalus puniceus venom from Malaysia (Lee et al., 2020; Liew et al., 2020), where GPVAV was shown to have 1.4 times more neutralization potential than Myanmar Russell's viper antivenom in neutralizing Myanmar green pit viper Trimeresurus erythrurus (Yee et al., 2020). In India, polyvalent antivenom (PAV) raised with an immunization mixture containing a venom pool of Naja naja, Daboia russelii, Echis carinatus and Bungarus caeruleus (the “Big Four” species) is used for treatment of green pit viper envenomation as it is the only available antivenom. Although PAV is largely effective against envenomation caused by the Big Four, various studies have revealed its poor performance in neutralizing venom toxins of these snakes from different geographical locations (Ali et al., 2013; Deka et al., 2019a; Faiz et al., 2017a). The inefficacy of antivenom in neutralizing venom of snake species from certain geographical locations is attributed to venom variation at both inter- and intra-specific levels (Chippaux et al., 1991). Therefore, assessment of efficacy of PAV in neutralizing venom toxins as well as understanding molecular characteristics and venom variability of region-specific medically important snakes is of utmost significance.
In India, among the green pit vipers, only the venom of Craspedocephalus malabaricus, endemic to the Western Ghats, has been studied well for pharmacological profiling as well as identification and isolation of functional peptides. Gowda and his team from University of Mysore, Karnataka isolated three different venom proteins: Trimarin (Kumar et al., 2010) and Malabarin (D Raghavendra Gowda et al., 2011) belonging to the metalloprotease family, and Malabarase (Kumar et al., 2013) belonging to the serine protease family. Immunoreactivity assays of C. malabaricus venom towards Indian Polyvalent Antivenom showed weak neutralization potential (Vanuopadath et al., 2020). However, the common green pit vipers inhabiting the eastern/north-eastern and Himalayan region, Trimeresurus erythrurus (Spot-tailed pit viper), Viridovipera medoensis (Medo pit viper), Popeia popeiorum (Pope's pit viper) and Trimeresurus septentrionalis (Northern white-lipped pit viper), which have similarities with species in South-east Asia, have remained unstudied (Fig. 1). Previously, Mitrakul (1973) reported Trimeresurus erythrurus and Popeia popeiorum venoms to possess clotting activity by thrombin-like action, fibrinolytic activity and direct aggregating effect on platelets (Mitrakul, 1973), but this has not been followed by studies regarding crude venom profiling, cross-reactivity with antivenom or isolation of peptides/proteins to understand structure-function. In the present study, the crude venom profiling of several green pit viper species from Mizoram, Himachal Pradesh and Arunachal Pradesh was carried out to understand their variation in terms of biochemical activities. Moreover, immunological cross-reactivity with Indian polyvalent antivenom as well as Thai green pit viper antivenom was also studied. A clinical case study based on 100 patients with confirmed green pit viper bites was also analysed with reference to the treatment protocols and clinical effects of commercially available Indian Polyvalent Antivenom administration on patients.
Section snippets
Venoms and antivenoms
Venom samples were collected from four different species of green pit vipers under permit from three geographical locations of India: Mizoram (Popiea popieorum and Trimeresurus erythrurus) and Arunachal Pradesh (Viridovipera medoensis) in northeast India and Himachal Pradesh (Trimeresurus septentrionalis) in northern India. Authentication of snakes was done by DNA analysis of mitochondrial genes (cytb, ND4, 12S and 16S, reported elsewhere) and the venom of one individual per species was
Comparative analysis of SDS-PAGE profile of venoms
The electrophoretic profiles of the crude venoms, analysed by gel electrophoresis under reducing conditions, revealed that the majority of protein bands were in the molecular weight range of 15–72 kDa in all the species studied (Fig. 2). Markedly, 4–5 clusters of protein bands at approximately 15 kDa, 30–37 kDa, 50–55 kDa, and 60–75 kDa can be seen in all the lanes, which broadly gives an idea of presence of major snake venom protein families such as phospholipase A2 (PLA2), snake venom serine
Conclusion
The present study unveils the biochemical profile of green pit vipers prevalent in northern and north-eastern part of India, correlating it with various pathophysiological manifestations of envenomation by these species. Comparison of in vitro biochemical activities between these species demonstrates Trimeresurus septentrionalis venom to exhibit highest activity in maximum number of biochemical activities studied. The para-specific green pit viper antivenom (GPVAV) shows good neutralization
Author contribution
ST: Methodology, Validation, Formal analysis, Investigation, Writing-original draft, RD: Conceptualization, Methodology, Validation, Resources, Writing-review and editing, Supervision, AM: Resources, Writing-review and editing, SG: Clinical Data, HTL, OM, VS, GM: Resources.
Ethical statement
No human or animal subjects were involved with the study.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to thank the National Biodiversity Authority of India, the Forest Departments of Himachal Pradesh, Mizoram, West Bengal, Assam, and Arunachal Pradesh for permission to carry out the sampling under permit numbers NBA/9/1673/17/18–19/3831, FFE-FB-F(10)-3/2017, A.33011/5/2011-CWLW/305, and CWL/G/13(95)/2011-12/PT.IV/respectively. Funding for this work was provided by the European Union Seventh Framework Programme (grant agreement number PIRSES-GA-2013-612131) to the BITES consortium
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