Proteomics and neutralization of Bungarus multicinctus (Many-banded Krait) venom: Intra-specific comparisons between specimens from China and Taiwan

Highlights

• Variations in Many-banded Krait (Bungarus multicinctus) venom were studied.

• Specimens were from mainland China (BM-China) and insular Taiwan (BM-Taiwan).

• Proteomes, antigenicity and neutralization of the venoms were examined.

• BM-China and BM-Taiwan venoms have similar major protein composition.

• Antivenoms produced in China and Taiwan were effective in neutralizing both venoms.

Abstract

The Many-banded Krait (Bungarus multicinctus) is a medically important venomous snake in East Asia. This study investigated the venom proteomes of B. multicinctus from Guangdong, southern China (BM-China) and insular Taiwan (BM-Taiwan), and the neutralization activities of two antivenom products (produced separately in China and Taiwan) against the lethal effect of the venoms. The venom proteomes of both specimens contained similar toxin families, notwithstanding small variations in the subtypes and abundances of minor components. More than 90% of the total venom proteins belong to three-finger toxins (3FTx, including alpha-neurotoxins) and phospholipases A₂ (PLA₂, including beta-bungarotoxins), supporting their key involvement in the pathophysiology of krait envenomation which manifests as pre- and post-synaptic neurotoxicity. The venoms exhibited potent neurotoxic and lethal effects with extremely low i.v. LD₅₀ of 0.027 μg/g (Bm-China) and 0.087 μg/g (Bm-Taiwan), respectively, in mice. Bungarus multicinctus monovalent antivenom (BMMAV) produced in China and Neuro bivalent antivenom (NBAV) produced in Taiwan were immunoreactive toward both venoms and their toxin fractions. The antivenoms neutralized the venom lethality variably, with BMMAV being more efficacious than NBAV by approximately two-fold. Findings suggest that the monovalent antivenom has a higher potency presumably due to its species-specificity toward the krait venom.

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.