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

Highlights

• Green pit vipers from north-east and northern India showed considerable differences in venom profiles.

• Variation in biochemical activities causing pathophysiological effects were observed.

• Thai green pit viper monovalent antivenom showed better neutralization potency than Indian Polyvalent Antivenom.

Abstract

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.

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.