Biogeographical venom variation in the Indian spectacled cobra (Naja naja) underscores the pressing need for pan-India efficacious snakebite therapy

Abstract

Background: Snake venom composition is dictated by various ecological and environmental factors, and can exhibit dramatic variation across geographically disparate populations of the same species. This molecular diversity can undermine the efficacy of snakebite treatments, as antivenoms produced against venom from one population may fail to neutralise others. India is the world's snakebite hotspot, with 58,000 fatalities and 140,000 morbidities occurring annually. Spectacled cobra (Naja naja) and Russell's viper (Daboia russelii) are known to cause the majority of these envenomations, in part due to their near country-wide distributions. However, the impact of differing ecologies and environment on their venom compositions has not been comprehensively studied.

Methods: Here, we used a multi-disciplinary approach consisting of venom proteomics, biochemical and pharmacological analyses, and in vivo research to comparatively analyse N. naja venoms across a broad region (>6000 km; seven populations) covering India's six distinct biogeographical zones.

Findings: By generating the most comprehensive pan-Indian proteomic and toxicity profiles to date, we unveil considerable differences in the composition, pharmacological effects and potencies of geographically-distinct venoms from this species and, through the use of immunological assays and preclinical experiments, demonstrate alarming repercussions on antivenom therapy. We find that commercially-available antivenom fails to effectively neutralise envenomations by the pan-Indian populations of N. naja, including a complete lack of neutralisation against the desert Naja population.

Conclusion: Our findings highlight the significant influence of ecology and environment on snake venom composition and potency, and stress the pressing need to innovate pan-India effective antivenoms to safeguard the lives, limbs and livelihoods of the country's 200,000 annual snakebite victims.

Fig 1. Sampling locations and SDS-PAGE profiles of N. naja venoms from distinct biogeographic zones of India.

This figure depicts (A) the venom sampling locations across distinct biogeographic zones of India and (B) SDS-PAGE profiles of N. naja venoms under reducing conditions. M: Protein marker (units in kDa); PB: Punjab; TN: Tamil Nadu; AP: Andhra Pradesh; WB: West Bengal; RJ: Rajasthan; MH: Maharashtra; and MP: Madhya Pradesh. The map of India shown here was prepared with QGIS 3.8 [43].

Fig 2. Biogeographic venom variability in N. naja.

HPLC profiles of N. naja venoms from various biogeographic zones of India are depicted here. A plot of absorbance values (mAU) at 215 nm against retention time (min) highlights the dramatic variation in the pan-Indian populations of this species. The doughnut charts are based on the area under the curve of the respective fractions (uniquely encoded with colours and numbers).

Fig 3. Proteomic compositions of N. naja venoms from various biogeographic regions.

Doughnut charts depicting the relative abundances of various toxins comprising the venoms of N. naja are presented here. Individual toxins are colour coded, and their relative abundances are indicated in percentages.

Fig 4. Pan-Indian N. naja venom-induced coagulopathies.

The abilities of venoms of various populations of N. naja to cause perturbations to the blood coagulation cascade via extrinsic (A) and intrinsic (B) pathways are depicted here as heatmaps. Numbers inside cells indicate the time (sec) required for the formation of the first fibrin clot. A colour key representing time in sec is also provided for each heatmap. Haemolytic activities of N. naja (C) venoms, defined as the percentage relative activity of the positive control (0.5% Triton X), are also shown.

Fig 5. The immunological cross-reactivity of commercial Indian antivenoms against Naja venoms.

Quantification of antibody binding of various commercial Indian antivenoms and naive horse IgG to the various snake venoms, determined by ELISA. Absorbance was measured at 405 nm for various dilutions (1:500, 1:2500 and 1:12500) of the antivenom, and the extent of binding shown as a colour gradient from red (low binding) to blue (high binding).

Fig 6. Toxicity profiles of N. naja from various biogeographic zones across India, and the neutralisation potencies of commercial Indian antivenom against these venoms.

Murine intravenous median lethal doses (expressed in mg/kg) of various populations of N. naja venoms (A) and the neutralising potencies (expressed in mg/ml) of the Premium Serums commercial antivenom against these venoms (B). The vertical dotted lines in panel B indicate the marketed neutralising potency (0.60 mg/ml) of commercial antivenoms against the N. naja venom.

Fig 7. The negative impact of biogeographic venom variability on Indian snakebite therapy.

This figure depicts the repercussions of biogeographic venom variability on snakebite treatment in India. Antivenom vials indicate the relative differences in neutralisation potencies against the geographically distinct populations of N. naja (yellow), and B. caeruleus (purple) in comparison to the source population in southern India, where the red dotted line on the vials represents the marketed neutralising potency of commercial Indian antivenoms. The intensity of purple clouds on the map is indicative of the estimated standardised snakebite death rates per million reported by Suraweera et al. 2020 [6], where the brighter regions represent the major hotspots. Geographical locales are defined by the box in the top right. The map of India shown here was prepared with QGIS 3.8 [43].