Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
Proteomics and neutralization of Bungarus multicinctus (Many-banded Krait) venom: Intra-specific comparisons between specimens from China and Taiwan
Graphical abstract
Introduction
Snakebite envenomation is a neglected tropical disease, affecting many impoverished and geo-politically marginalized populations in the tropics and subtropics (Chippaux, 2017; Gutierrez et al., 2017). Each year, approximately 5.4 million snakebites occur, resulting in 1.8 to 2.7 million cases of envenomation, from which 81,000 to 138,000 deaths and three times as many amputations and other permanent disabilities ensue (Kasturiratne et al., 2008; WHO, 2016). In Asia, snakebite is prevalent, and the heavy burden of snakebite envenomation commonly falls on the rural populations who are engaged in agricultural activities, including those in the mainland China and the island of Taiwan (Kasturiratne et al., 2008; Mao et al., 2017; Qin, 1998; WHO, 2016). Separated by the Taiwan Strait, the Pacific subtropical island of Taiwan shares similar herpetofauna with southern China; these include elapids such as the Many-banded Krait (Bungarus multicinctus) and Chinese Cobra (Naja atra), and viperids such as the habu (Protobothrops mucrosquamatus), Hundred-pacer or Five-pacer (Deinagkistrodon acutus) and Eastern Russell's Viper (Daboia siamensis) (Ming-Yi and Ruey-Jen, 2008), all of which are medically important and capable of inflicting fatal envenomation.
Among the elapids, kraits (Genus: Bungarus) are broadly distributed in Asia, and the envenomations caused by most krait species are highly fatal (Kuch et al., 2011; Pillai et al., 2012; Sarkar et al., 2018; Tongpoo et al., 2018). Krait envenomation does not cause significant local reactions typically but can cause severe systemic paralysis that leads to respiratory failure and death if proper treatment is unavailable. In East Asia, B. multicinctus has been reported to cause approximately 8% and 7.5% of total snakebite cases in China (Qin, 1998) and Taiwan (Mao et al., 2017), respectively. Due to its wide occurrence and severe outcome of envenomation, B. multicinctus is duly classified as a Category 1 medically important venomous snake species in the East Asia by the World Health Organization (WHO, 2016). In contrast to cobra (Naja atra) envenomation, B. multicinctus bites are associated with a higher mortality rate with delayed neurological manifestation, without severe local envenoming effects such as inflammation (pain, swelling) and skin necrosis (Mao et al., 2017; Pe et al., 1997). Consequently, the bite might be overlooked, or the victims could be misdiagnosed as having been bitten by a non-venomous snake and hence delayed in receiving appropriate treatment. The neurotoxic effect of krait envenomation is often described as “resistant neurotoxicity”, attributed to nerve terminal damage that depletes neurotransmitter (acetylcholine) at the neuromuscular junction, and the regeneration of the synapses can take days (Hung et al., 2010; Ranawaka et al., 2013).
The venom of B. multicinctus has been extensively studied for decades, and its principal toxins, i.e., the beta-bungarotoxins (β-BTx) and alpha-bungarotoxins (α-BTx), were among the most well characterized snake toxins (Kondo et al., 1982; Mebs et al., 1972). The complexity of B. multicinctus venom composition, however, was only revealed much more recently with the advent of proteomics, reported for specimens from Vietnam (Vinh Phuc Province) (Ziganshin et al., 2015), China (Guangxi) (Shan et al., 2016) and Taiwan (Liu et al., 2018). Different methods of analysis were applied, and the proteomic findings were, by and large, variable among the studies, in particular on the abundances and subtypes of proteins in the different venom samples. Comparison and further interpretation of intra-specific variation of B. multicinctus venom are, therefore, challenging. From the medical perspective, it is important to address intra-specific venom variability, since this is often the major event that contributes to variable toxic manifestation and discrepancy in antivenom efficacy (Casewell et al., 2020; Chaisakul et al., 2019; Hia et al., 2020; Sharma et al., 2014; Tan et al., 2020). Causes of intra-specific venom variation are many, with geographical factors being the most commonly cited (Tan et al., 2020; Tan et al., 2015; Wong et al., 2018). In this context, venom variation in B. multicinctus venom is anticipated. B. multicinctus native to Taiwan represents the most eastern dispersal of krait species, while the Strait of Taiwan has long segregated the insular population from those on the mainland (southern China), restricting recent genetic exchange between them. In China, a monovalent antivenom specific for the Chinese B. multicinctus venom (BMMAV) is produced by Shanghai Serum Biological Technology Co., Ltd., China; whereas in Taiwan, a Neuro bivalent antivenom (NBAV) raised against B. multicinctus and N. atra venoms of Taiwanese origin was produced by the Centres for Disease Control, Taiwan. The efficacy of the two antivenoms in neutralizing the venom of the corresponding native B. multicinctus, and that from the other geographical population across the Strait, has not been examined and compared in depth. We hypothesized that small variations exist in the proteome, toxicity and antigenicity of B. multicinctus venoms from the two locales, and the monovalent antivenom (produced in China) is likely to be more potent than the bivalent antivenom (produced in Taiwan) due to its mono-specific nature toward the krait venom. The study thus investigated the geographical variation and neutralization of B. multicinctus venom, and to provide insight into the geographical utility of antivenom product. Effective antivenoms can be better distributed and utilized in areas where locally manufactured antivenoms are inadequately supplied.
Section snippets
Venoms and antivenoms
The venom samples of B. multicinctus from Taiwan Island (Bm-Taiwan) and Guangdong of southern China (Bm-China) were, respectively, supplied by Latoxan Serpentrium (France) and a local market supplying snakes in Guangzhou, China. The Calloselasma rhodostoma venom from Java (Latoxan, France) was used as negative control in the indirect enzyme-linked immunosorbent assay (ELISA). All venoms were lyophilized and stored at −20 °C until use.
Two antivenom products used in this study were: (a) Bungarus
Chromatographic and electrophoretic profiling of B. multicinctus venoms
The venoms of B. multicinctus from China (Bm-China) and Taiwan (Bm-Taiwan) were fractionated by C18 RP-HPLC under the same experimental settings. The chromatograms showed similar patterns of protein elution over 180 min for both venoms, yielding 16 major fractions each (Fig. 1). The SDS-PAGE profiles of Bm-China and Bm-Taiwan protein fractions (F1-F16) were also similar (Fig. 1). The electrophoretic profiles showed that both venoms were comprised mainly of low molecular weight proteins
Discussion
Bungarus multicinctus is a common medically important venomous snake species in East Asia, mainly distributed in the central, southern region of mainland China and in Taiwan. The present study showed that the venoms of Bungarus multicinctus from Guangdong, southern China (Bm-China) and insular Taiwan (Bm-Taiwan) have similar protein composition. In comparison to previous proteomic studies, 3FTx and PLA2 consistently formed the major toxin families in B. multicinctus venoms sourced from
Conclusion
The venoms of B. multicinctus from China and Taiwan showed a comparable proteomic profile that is characterized by abundant 3FTx (alpha-bungarotoxins and kappa-bungarotoxins) and PLA2 (beta-bungarotoxins). These proteins are collectively the major principal toxins in the venom that account for the potent neurotoxicity and fatality of B. multicinctus envenomation. BMMAV and NBAV, two antivenom products available in the region, were able to immunorecognize the principal toxins and neutralize the
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.
Acknowledgment
Authors are grateful for the research funding from the University of Malaya (BKS003-2020 and ST032-2019).
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2023, International Journal of Biological MacromoleculesAn immunodetection assay developed using cobra cytotoxin-specific antibodies: Potential diagnostics for cobra envenoming
2022, ToxiconCitation Excerpt :The use of CTX-specific antibodies is justified by the fact that all cobra venoms virtually contain cytotoxins (∼16–75% of total venom proteins) which can serve as the antigenic analytes for immunodetection of cobra venoms (Lauridsen et al., 2017; Palasuberniam et al., 2021; Petras et al., 2011; Tan et al., 2019c; Tan et al., 2015c; Tan et al., 2020; Tan et al., 2017c; Wong et al., 2018; Yap et al., 2014b). Meanwhile, CTX is absent from the venoms of other medically important snake species in the region, including vipers and pit vipers (Lee et al., 2021; Liew et al., 2020; Lingam et al., 2020; Tan et al., 2021a; Tan et al., 2019a; Tan et al., 2017a; Tang et al., 2016) as well as the other neurotoxic elapid snakes, e.g., kraits and sea snakes (Chapeaurouge et al., 2018; Oh et al., 2017; Oh et al., 2019; Oh et al., 2021; Tan et al., 2015b; Tan et al., 2019b; Tan et al., 2017b; Ziganshin et al., 2015). Although CTX has been found in King Cobra venom in the form of beta-cardiotoxin (Chang et al., 2013; Lertwanakarn et al., 2020; Petras et al., 2015; Tan et al., 2015a), the amount is typically lower (0.4–9%) than its presence in cobra venoms.
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2022, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsCitation Excerpt :There are at least 16 extant Bungarus species throughout Asia, and of these, at least three are designated as highly medically important venomous snakes of Category 1 by the World Health Organization, for their ability to cause significant snakebite problem in the respective continents: Bungarus caeruleus in South Asia, Bungarus candidus in Southeast Asia, and Bungarus multicinctus in East Asia (WHO, 2016a; Tan et al., 2021b). The venom properties of these major kraits have been long and extensively studied (Shan et al., 2016; Silva et al., 2016; Oh et al., 2017; Rusmili et al., 2019; Oh et al., 2021), while research is lacking for many lesser-known Bungarus species. Of these, the Red-headed Krait (Bungarus flaviceps) is an endemic species in Southeast Asia, distributed in parts of Indochina, Peninsular Malaysia, Borneo, and some Indonesian islands (Bangka, Sumatra, Belitun) (Uetz and Hosek, 2022) (see graphical abstract).
Sharp-nosed Pit Viper (Deinagkistrodon acutus) from Taiwan and China: A comparative study on venom toxicity and neutralization by two specific antivenoms across the Strait
2022, Acta TropicaCitation Excerpt :Geographically, the Taiwan Strait, a 180-kilometer-wide sea barrier completely separates the island of Taiwan from the continental Asia since the Pleistocene glaciation period. The long ecological separation of D. acutus populations across the Strait may have an impact on the venom properties, where intra-species variation might exist as seen in a number of venomous snake species with populations separated by sea (Lingam et al., 2020; Oh et al., 2021; Tan et al., 2018). The understanding of intra-species venom variability is vitally important as it has consequences on the venom toxicity, and often compromises the treatment outcome especially when the antivenom is raised against snakes from a geographically distant population albeit being the same species (Faisal et al., 2018; Oh et al., 2017; Pla et al., 2019).
Varespladib (LY315920) rescued mice from fatal neurotoxicity caused by venoms of five major Asiatic kraits (Bungarus spp.) in an experimental envenoming and rescue model
2022, Acta TropicaCitation Excerpt :Venomous snakes in Category 2 are species with limited epidemiological or clinical data pertaining to envenoming, or, they are less frequently implicated due to their activity cycles, behavior, habitat preferences or occurrence in areas remote to large human populations (WHO, 2016). Although antivenom products indicated for krait bite envenomings are largely available in the various regions, the neutralization potencies of the products against krait venom-induced lethality were considerably low (potencies <1 mg/ml, amount of venom neutralized completely per volume unit of antivenom), and cross-species efficacy is even limited (Fig. 1) (Oh et al., 2017, 2019, 2021). The development of next-generation antivenom with either toxin-specific pan-species antivenoms or synthetic antibodies (Laustsen et al., 2018; Ratanabanangkoon et al., 2016, 2020) is a feasible solution but requires enormous time and cost to reach clinical application.