Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization

Abstract

Abstract. Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cationexchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography-mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A 2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03-0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 μL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.

Comparative venom gland transcriptomics ofNaja kaouthia(monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

“…The acidic Asp-49-PLA 2 , however, was not lethal in mice even at a dose of >10–20 times of the median lethal dose of whole venom (Tan et al, 2016a). This is consistent with the report of non-toxic property of various acidic-type PLA 2 s isolated from the venoms of Indian N. kaouthia (Joubert & Taljaard, 1980) and Pakistani N. naja (Wong, Tan & Tan, 2016), but diverged markedly from the highly lethal, neutral/basic venom PLA 2 s characterised for other Southeast Asian cobras including Naja sumatrana and N. sputatrix (Leong et al, 2015; Tan & Arunmozhiarasi, 1989). The acidic PLA 2 s hence may serve a secondary role of ancillary function, for instance, potentiating the toxic actions of other venom components, including CTXs/cardiotoxins (Gasanov, Dagda & Rael, 2014), SVMPs (Bustillo et al, 2015) and weak neurotoxin (Mukherjee, 2010) to enhance tissue damages which are crucial for prey digestion but clinically deleterious as it complicates local tissue necrosis.…”

Revisiting Notechis scutatus venom: on shotgun proteomics and neutralization by the “bivalent” Sea Snake Antivenom

“…By incorporating N. scutatus venom in the immunogen, the neutralization of H. schistosus venom by SSAV is likely improved with the additional antibody titer raised against its myotoxic, basic PLA 2 s; however, the overall potency of the antivenom appears to remain limited by the weak neutralization of short neurotoxins which are present in a very large amount in sea snake venom [15]. This is in agreement with recent reports on the consistently weak neutralization of antivenoms against short neurotoxins isolated from Asiatic cobra venoms (Naja naja, Naja kaouthia and Naja sputatrix) [37][38][39]. On the other hand, the neutralization of N. scutatus venom is attributable primarily to SSAV neutralizing the abundant PLA 2 s, and enhanced by the additional anti-titers against the coagulant enzymes and alpha-neurotoxins.…”

Assessing SABU (Serum Anti Bisa Ular), the sole Indonesian antivenom: A proteomic analysis and neutralization efficacy study

“…This is in agreement with the higher content of alpha-neurotoxins in N. sumatrana venom compared to N. sputatrix venom, which has a much higher content of cytotoxins26. The LD 50 value of cobra cytotoxin is about 10-fold higher (hence less lethal) than the alpha-neurotoxin when administered intravenously92728. On the other hand, krait venoms are known to exhibit high lethality (LD 50  < 0.2 μg/g, including Indonesian B. candidus ), except for B. fasciatus of Thailand which has an exceptionally higher venom LD 50 exceeding 1.0 μg/g1829.…”

“…The inadequate efficacy of SABU could be due to the presence of other “junk proteins” and non-specific F(ab’) 2 that do not react with the toxin antigens concerned. On the other hand, while antivenom neutralization against cobra and krait toxins (especially the low molecular mass alpha-neurotoxins) is known to be limited928, such “weakness” of antivenom is in particular serious for SABU, judging from its feeble neutralization against the venoms of N. sputatrix and B. candidus , two medically important species in Java - the most densely inhabited island of Indonesia. From the practical standpoint, the findings indicate the urgency for a revised manufacturing process to ensure the production of a safe and effective antivenom in the country, where it should be furnished with the qualities of improved purity, higher potency and a wider coverage of species in Indonesia.…”

“…These parameters were determined according to the probit analysis method of Finney42 using BioStat 2009 analysis software (AnalystSoft Inc., Canada). The neutralization capacity was also expressed in term of ‘neutralization potency’ (P, defined as the amount of venom neutralized completely by a unit of antivenom) according to the recommendation by Morais et al 2843…”