Biochemical and Biological Profiles of Bangladeshi Russell's Viper Snake Venom and Neutralizing Efficacy by Indian VINS Polyvalent Antivenom

Abstract

The Russell's viper (Daboia russelii) has recently become a significant threat to human life in Bangladesh. Given its wide distribution across South Asia, the venom characteristics and lethality can vary by region with different toxicological properties. Hence, we investigated the characteristics of Bangladeshi Russell's viper venom (BRVV) through SDS-PAGE profiling, reverse-phase HPLC analysis, along with assessments of phospholipase A₂ (PLA₂), edema-inducing, hemolytic, hemorrhagic, and coagulant activities, histopathology, and blood biochemistry, following established protocols. We also studied the neutralization efficacy of polyvalent antivenom from VINS Bio Products Ltd., India (VPAV) against BRVV. RP-HPLC analysis of BRVV displayed 15 peaks, and SDS-PAGE showed high-intensity protein bands within the 15-70 kDa range. The median lethal dose (LD₅₀) for mice was found to be 0.33 mg/kg intraperitoneally (i.p.), and venom exposure resulted in neurotoxic symptoms such as limb paralysis, respiratory difficulties, and sluggishness. BRVV exhibited strong PLA₂, procoagulant, hemorrhagic, indirect hemolytic, and edema-inducing activities but poor direct hemolytic activity. Venom administration also significantly increased levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), cholesterol, total protein, uric acid, blood urea nitrogen (BUN), and creatinine in mouse serum, indicating organ damage. Histopathological examination revealed cell vacuolization, congestion, hemorrhage, inflammatory infiltrations, and necrosis in venom-exposed tissues, validating the abnormal serum biochemistry. The neutralization study revealed that VPAV had limited efficacy against BRVV, suggesting the presence of venom proteins not fully neutralized by the antivenom. Altogether, these findings suggest that the Russell's viper is a medically significant venomous snake in Bangladesh, and VPAV is only partially effective in reducing the venom's toxic effects. Therefore, region-specific venoms must be considered in antivenom development for more effective treatment in envenomation cases.

Keywords: Bangladeshi Russell's viper; PLA2; VINS polyvalent antivenom; hemorrhage; histopathology; procoagulant.

Figure 1

SDS–PAGE electrophoresis of the crude venom on a 15% polyacrylamide gel under reducing conditions. Lane M: PageRuler plus protein ladders. The molecular weight of standard proteins is marked in kDa. Lanes C1 to C7: BRVV venom samples at 50 μg (C1), 75 μg (C2), 100 μg (C3), 125 μg (C4), 150 μg (C5), 175 μg (C6), and 200 μg (C7), respectively.

Figure 2

RP-HPLC analysis of BRVV. RP-HPLC was conducted on a discovery BIO wide pore C18 column pre-equilibrated with 0.1% (v/v) TFA. Approximately 3 mg of crude venom was added to the column and eluted with a linear gradient of 80% (v/v) ACN containing 0.1% (v/v) TFA over 56 min at a flow rate of 0.5 mL/min. The elution was monitored at 280 nm.

Figure 3

PLA₂ activity of BRVV against egg yolk suspension in the turbidometric method. The decreased turbidity of the suspension was recorded at 740 nm after incubation at 30°C and compared with the control when the different concentrations of venom hydrolyzed the phospholipids of egg yolk lipoproteins. Data are presented as the mean ± SD of three replicates.

Figure 4

Photograph showing the PLA₂ activity of BRVV against egg yolk solution in the agar plating method. Tris buffer represented no zone formation. A 5 μg venom dose induced a 13-mm transparent zone, while a 22-mm zone was developed by 30 μg venom.

Figure 5

The edema-inducing activity of BRVV for different venom concentrations. The edema-inducing activity is expressed in the edema ratio. All values are expressed as the mean ± SD (n = 3).

Figure 6

The procoagulant activity of BRVV on human plasma. The coagulation time of human citrated plasma was monitored in the presence of 200 mM of CaCl₂, and PBS clotting time was considered as normal clotting time. Each point represents the average ± SD of three independent experiments.

Figure 7

The hemorrhagic activity of Russell viper venom on the mouse skin. (a) The normal skin of control mice, and (b–e) hemorrhagic blood spots caused by various dosages of BRVV.

Figure 8

Histopathological sections of different organs in mice exposed to BRVV along with their respective controls, stained by eosin and hematoxylin. (a) In the control group, normal liver architecture was observed, including a clearly defined central vein (CV), organized hepatocyte cords (Hc), and visible Kupffer cells (Kc). (b) BRVV-treated groups exhibited pathological alterations such as central vein (^∗) congestion, localized areas of inflammatory cell infiltration (^∗∗), mild hemorrhage (Hr), cytoplasmic vacuolization (yellow arrow), karyorrhexis (arrowhead), karyolysis (arrow fork), and pyknotic nuclei (arrow). (c) Kidney sections from the control group displayed a normal renal cortex, characterized by intact glomeruli encircled by well-defined Bowman's space. (d) In the treated groups, the renal cortex exhibited pathological changes, including glomerular shrinkage and congestion (Cg), along with an abnormally widened Bowman's space (^∗), necrosis in glomeruli (arrowhead), tubular necrosis (arrow fork), vacuolization in epithelial cells (Vc), congestion (black star), and dilatation of tubule (Dt). (e) Control group demonstrated typical morphology with healthy mucosa (M), submucosa (SM), and muscularis mucosa (MM). (f) The BRVV-treated group exhibited degeneration of the intestinal crypts, accompanied by epithelial shedding of the intestinal glands into the lumen (^∗∗), degeneration of muscularis mucosa (black star), mild hemorrhage (Hr), and swelling and destruction of mucosal area (black arrowhead). (g) The untreated heart tissues show a normal appearance of cardiac muscle. (h) The venom-treated groups depict disorganization of myofibrils with the destruction of muscular striation in different areas, blood congested area (black star), cytoplasmic vacuolization (yellow arrow), and ruptured muscle fibers (yellow arrowhead).

Figure 9

Neutralization of PLA₂ activity of crude venom by VINS polyvalent antivenom in the turbidometric method. With the different amounts of antivenom, 1 μg of crude venoms was incubated at 37°C for 30 min, and then egg yolk solution was added. The increased turbidity of the suspension was recorded at 740 nm. Experiments were repeated thrice, and the mean values were used to plot the graph.

Figure 10

Inhibition of PLA₂ activity of BRVV by VINS polyvalent antivenom. Neutralization of PLA₂ activity of crude venom by VINS polyvalent antivenom was measured by the egg yolk agar plate method. Various antivenom concentrations (μg: μg) were applied to neutralize the minimum PLA₂ dose (MPD). All data are presented as the mean ± SD (n = 3). (a) Antivenom activity was measured based on the decrease in a clear zone (mm) at various venom: antivenom ratios (μg: μg), where the zone is caused by PLA₂ activity of venom. Decreased zone size indicates effective PLA₂ neutralization. (b) Corresponding percentage neutralization of PLA₂ activity by VINS antivenom.

Figure 11

In vitro neutralization of BRVV-induced coagulation by VINS polyvalent antivenom. VINS antivenom dissolved in PBS was incubated with the minimum coagulant dose (MCD) of BRVV for 30 min at 37°C. The mixture was then incubated with human plasma for 3 min at 37°C, followed by the addition of 40 μL of 200 mM CaCl₂ to initiate clotting. The data are shown as the mean ± SD (n = 3). Here, PBS = phosphate buffer saline and AV = antivenom. (a) Coagulation time (seconds) of BRVV-treated human plasma across different venom: antivenom ratios. Increased coagulation time indicates neutralization of the procoagulant effects of BRVV by VINS polyvalent antivenom. The control clotting time was determined using CaCl₂-activated plasma with PBS alone. (b) Corresponding percentage neutralization of BRVV-induced coagulation by VINS antivenom at the same venom: antivenom ratios.