Fig 1.
SDS-PAGE pattern, and zymography of H. hypnale, E. carinatus, and D. russelii venoms.
(A) SDS-PAGE (10%) banding pattern of venoms; Hhv, Ecv, and Drv (25 μg each) were used and analyzed under non-reducing condition. For zymography, the venoms Hhv, Ecv, and Drv were independently studied for proteolytic activity banding patterns in substrate gel assays. B-D and E-G respectively represent caseinolytic and gelatinolytic activities of three venoms. The substrates, casein, and gelatin (0.2%) were incorporated into respective gels (10%). The SDS-PAGE was performed under non-reduced condition. In lanes 1–4 in respective gels, different doses, 5, 10, 20, and 30 μg of venoms were used. M represents the molecular weight markers in kDa. The clear translucent zones against a blue background indicate the respective activities in gels. The images were captured by using HP Scanjet (Model-G2410).
Table 1.
Protease activity; one unit of activity is defined as the amount of enzyme required to cause an increase in O.D. by 0.01 at 660 nm/min (a).
Indirect hemolytic activity; it is expressed as a percent of hemolysis (b). Direct hemolytic activity; it is expressed as a percent of hemolysis (c). L-amino acid oxidase activity; one unit of activity is defined as the amount of enzyme required to cause an increase in O.D. by 0.001 at 440 nm/min (d). 5’-nucleotidase activity; one unit of activity is expressed in terms of the release of inorganic phosphorus in μ moles/min/μg of protein (e). The data is presented as Mean ± SEM (n = 3).
Fig 2.
Murine model of edema-inducing activity of H. hypnale, E. carinatus, and D. russelii venoms.
(A) Dose-dependent edema-inducing activity of Hhv, Ecv, and Drv. Different doses of venoms (1–5 μg) in 20 μl PBS were injected into the intraplantar surface of mice right footpads. The left footpads that received PBS alone were served as negative controls. After 1 h of injection, mice were euthanized using xylazine; both the legs were removed at the ankle joint and weighed. An increase in weight due to edema was calculated as the edema ratio, which equals the weight of the edematous leg × 100/weight of the negative control leg. (B) Mice foot pads showing hemorrhagic (Hhv and Ecv) and non-hemorrhagic (Drv) edema. The data is presented as Mean ± SEM (n = 3).
Fig 3.
Murine model of hemorrhagic activity of H. hypnale, E. carinatus, and D. russelii venoms.
(A) Groups of mice were independently injected intradermally with different doses, 1–5 μg of Hhv, and 5 μg each of Ecv and Drv in 30 μl PBS respectively. The group of mice were injected with PBS served as a negative control. After 3 h of injection, mice were euthanized using xylazine and removed skin at the venom injected spot, the area of hemorrhagic spots that appeared on the inner surface of the skin was measured in mm2. (B) The inner surface of the skin tissues shows hemorrhagic spots. The data is presented as Mean ± SEM (n = 3) and analyzed using one-way/two-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, and ns (not significant) >0.05. ‘*’ significant compared to the control group (PBS).
Fig 4.
Effect of H. hypnale, E. carinatus, and D. russelii venoms on coagulant activities.
(A) Plasma re-calcification time of Hhv; 200 μl of citrated human plasma was treated with 1–20 μg/ml of venom for 1 min at 37 0C, clotting was initiated by adding 20 μl 0.25 M CaCl2. (B) Hhv induced the plasma coagulation in the absence of CaCl2; clotting was initiated by adding 1–20 μg/ml of Hhv to the 200 μl of citrated human plasma. (C) Comparative plasma re-calcification time of Hhv, Ecv, and Drv; 200 μl of citrated human plasma was independently treated with 10 μg/ml each of venoms to initiate the clotting. For Drv, clotting was initiated separately by adding 20 μl 0.25 M CaCl2. (D) Activated partial thromboplastin time (APTT); 100 μl of citrated human plasma was treated independently with 5 μg/ml each of Hhv, Ecv, and Drv for 1 min at 37 0C, then 100 μl of reagent (LIQUICELIN-E phospholipids preparation derived from rabbit brain with ellagic acid) was added and incubated for 3 min at 37 0C. The clotting was initiated by adding 100 μl 0.02 M CaCl2. Prothrombin time (PT); 100 μl of citrated human plasma was treated independently with 5 μg/ml each of Hhv, Ecv, and Drv for 1 min at 37 0C. The clotting was initiated by adding 100 μl of PT reagent (UNIPLASTIN–rabbit brain thromboplastin). In all the cases, the plasma devoid of venom was served as control experiments. (E) Thrombin clotting time (TCT); 100 μl of fibrinogen (3 mg/ml) in 10 mM Tris-HCl buffer pH 7.6 was independently treated with 10 μg/ml each of Hhv, Ecv, and Drv to initiate the clotting. Thrombin, 10 U was used as a positive control, and fibrinogen alone was served as a negative control. In all the above experiments, the time taken for the visible clot formation was recorded in seconds. In all the cases, the data is presented as Mean ± SEM (n = 4) and analyzed using one-way/two-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, and ns (not significant) >0.05. ‘*’ significant compared to the control group (PBS).
Fig 5.
Fibrinogenolytic and fibrinolytic activities of H. hypnale, E. carinatus, and D. russelii venoms.
(A) Fibrinogenolytic activity of venoms (time-dependent); fibrinogen, 50 μg was independently incubated with 1 μg each of Hhv, Ecv, and Drv for different time intervals at 37 0C. Lane I fibrinogen (50 μg) alone, lanes 2–6 where 50 μg of fibrinogen was incubated with Hhv (Aa), Ecv (Ab), and Drv (Ac) for 5, 10, 15, 30, and 60 minutes respectively. (B) Fibrinogenolytic activity of venoms (dose-dependent); fibrinogen, 50 μg was independently incubated with different doses of Hhv, Ecv, and Drv for 60 min at 37 0C. Lane I fibrinogen (50 μg) alone, lanes 2–6 where 50 μg of fibrinogen was treated with 1, 2.5, 5, 7.5, and 10 μg of Hhv (Ba), Ecv (Bb), and Drv (Bc) respectively. (C) Fibrinolytic activity of venoms; 100 μl of washed plasma clot was independently incubated for 16 hours at 37 0C with 5 & 10 μg each of Hhv (lanes 2 and 3), Ecv (lanes 4 and 5), and Drv (lanes 6 and 7) respectively, and fibrin clot alone (lane 1). In all cases, M represents the molecular weight protein markers in kDa and are analyzed on SDS PAGE (10% for fibrinogenolytic activity and 7.5% for fibrinolytic activity) under reduced condition. The images were captured by using HP Scanjet (Model-G2410). The quantitative degradation patterns of fibrinogen, and fibrin by the Hhv, Ecv, and Drv are represented through corresponding densitogram images (ImageJ Software Ver. 1.53k, USA) in respective cases.
Fig 6.
Murine model of myotoxicity and cardiotoxicity of H. hypnale, E. carinatus, and D. russelii venoms.
The groups (n = 4) of mice were independently injected intramuscularly (i.m.) with the LD50 doses of Hhv (3.5 mg/kg), Ecv (2.5 mg/kg), and Drv (3.0 mg/kg) into the thigh muscle in 50 μl of PBS. The groups of mice that received PBS alone served as control experiments. After 24 hours, mice were anesthetized using xylazine; blood was drawn by cardiac puncture. The marker enzymes, LDH (A), CK (B), and CK-MB (C) activities (Units/L) were assayed in the mice serum. The data is presented as Mean ± SEM (n = 4) and analyzed using one-way/two-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, and ns (not significant)>0.05. ‘*’ significant compared to the control group (PBS).
Fig 7.
Cross-reactivity of H. hypnale, E. carinatus, and D. russelii venoms with the anti-venoms.
(A) SDS-PAGE protein banding pattern of Hhv, Ecv, and Drv, and M represents the molecular weight protein markers in kDa. (B) Western blots showing reactivity/cross-reactivity of HhAV (Ba), EcAV (Bb), DrAV (Bc), BhAV (Bd), ViAV (Be), and pre-immune rabbit serum (Bf) with Hhv, Ecv, and Drv. BSA, 25 μg was used as a negative control. (C) Corresponding Ponceau-S stain images showing the protein bands in respective membranes when stained before processing with respective anti-venoms in Western blot. In all cases, the electrophoresis was carried out under identical, non-reduced condition in 10% gels. (D) Indirect ELISA titers of Hhv, Ecv, and Drv with anti-venoms. (Da) Hhv, (Db) Ecv, and (Dc) Drv. Venoms, 0.1 μg/100 μl were independently treated with various dilutions of HhAV, EcAV, DrAV, BhAV, and ViAV. The data is presented as Mean ± SEM (n = 4). (Antiserums, 1 mg/ml were adjusted, and used for dilution in ELISA).
Fig 8.
Neutralization of biochemical activities of H. hypnale venom by anti-venoms.
(A) Proteolytic, (B) Indirect hemolytic, (C) L-Amino acid oxidase, and (D) 5’-Nucleotidase activities. Hhv was independently pre-incubated with various doses (50–10000 μg/ml) of HhAV, EcAV, DrAV, BhAV, and ViAV for 15 min at room temperature. Protease activity; 50 μg/ml of Hhv alone was considered as 100% activity, hemolytic activity; 20 μg/ml of Hhv alone was considered as 100% activity, L-amino acid oxidase activity; 100 μg/ml of Hhv alone was considered as 100% activity, and 5’-Nucleotidase activity; 15 μg/ml of Hhv alone was considered as 100% activity. The data is presented as Mean ± SEM (n = 3).
Fig 9.
Neutralization of edema inducing activity and hemorrhagic activity of H. hypnale venom by anti-venoms using murine model.
(A) Edema inducing activity, and (B) hemorrhagic activity of Hhv; in both cases, Hhv was independently pre-incubated with various doses (50–500 μg) of HhAV, EcAV, DrAV, BhAV, and ViAV for 15 min at room temperature. Hhv, 5 μg alone was considered as 200% edema-inducing activity, and 5 μg Hhv alone was considered as 100% hemorrhagic activity. The data is presented as Mean ± SEM (n = 3).
Fig 10.
Neutralization of H. hypnale venom lethality by anti-venoms using the murine model.
Groups (n = 6) of mice were independently injected intraperitoneally (i.p.) with 2 LD50 (7 mg/kg) dose of Hhv in 50 μl of PBS. Post 10 min of venom injection, the mice were administered with ED50 (140 mg/kg), and 2 ED50 (280 mg/kg) doses of HhAV, and 700 mg/kg body weight of EcAV, DrAV, BhAV, and ViAV independently via tail vein (i.v.). Groups of mice that received venom alone and PBS were served as control experiments. Mice were kept under observation for 24 h and the time of death was recorded. The percent survival analysis of mice was done by constructing the Kaplan-Meier survival curve, the p-value was calculated using the log-rank (Mantel-Cox) test, ***p < 0.001, and **** p < 0.0001.
Fig 11.
Neutralization of H. hypnale venom-induced myotoxicity and cardiotoxicity by anti-venoms using the murine model.
Groups (n = 4) of mice were independently injected intramuscularly (i.m.) with LD50 dose (3.5 mg/kg) of Hhv in 50 μl of PBS. Post 10 min of venom injection, the mice were administered with ED50 (140 mg/kg) dose of HhAV and 700 mg/kg doses of EcAV, DrAV, BhAV, and ViAV independently via tail vein (i.v.). The groups of mice that received PBS alone were served as control experiments. After 24 h, mice were anesthetized using xylazine; blood was drawn by cardiac puncture. The marker enzymes, LDH (A), CK (B), and CK-MB (C) activities (Units/L) were assayed in serum. The data is presented as Mean ± SEM (n = 4) and analyzed using one-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, and ns (not significant) p >0.05. ‘*’ significant compared to Hhv + HhAV.
Table 2.
Neutralization of coagulant activity (Plasma re-calcification time, APTT, and PT assays), and thrombin-like activity of H. hypnale venom by anti-venoms: Hhv was independently pre-incubated with various doses (100–2000 μg/ml) of HhAV, EcAV, DrAV, BhAV, and ViAV for 15 min at room temperature.
In all cases, 20 μg/ml of Hhv alone was considered as 100% coagulant activity and thrombin-like activity. The data is presented as Mean ± SEM (n = 4) and analyzed using one-way ANOVA followed by Bonferroni post-tests, ‘**** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05, and ns (not significant) p >0.05. ‘*’ significant compared to the control group (PBS). ‘*’ significant compared to Hhv+ HhAV.