Fig 1.
Clinical features of myotoxicity in 245 confirmed Sri Lankan Russell’s viper envenomings (a), the presence of localised and generalised myalgia over time; (b), the presence of localised and generalised muscle tenderness over time. Note: data of all 245 patients were not available at any given time because some patients were admitted later than the time point or discharged before the time point, or the clinical assessment of the patient was not possible at that time point because the patient was sedated, unconscious or transported away for investigations.
Table 1.
Clinical and epidemiological data of the 245 patients recruited for the present study.
Fig 2.
Serum creatine kinase (CK) concentrations in 219 study participants: (a) Scatter plot comparing the CK concentrations (at 24 h post bite) of patients with no features, patients with features of local myotoxicity (myalgia or tenderness) and patients with features of systemic myotoxicity (p = 0.049; Kruskal-Wallis test); (b), peak serum concentrations of CK versus venom in 37 patients who had CK >300U/l at 24 h post-bite (Spearman’s correlation; p = 0.48); (c), CK concentration at 24 h versus time from the snakebite to the first antivenom dose in 178 patients who received antivenom (note: 12 patients who received antivenom had no CK due to the unavailability of the sample at 24 h); (d), plots of the CK concentrations versus time since bite for the 37 patients; (e), plots of the venom concentrations versus time for the 37 patients.
Fig 3.
Chromatograms of Sri Lankan Russell’s viper venom and fractions (a), Size-exclusion chromatogram of the whole venom on Superdex G75 column; (b), Reverse Phase High Performance Liquid Chromatogram (RP-HPLC) of the fraction ‘E’ on Jupiter C18 semi-preparative column. Note: the peaks eluting at 31 and 38 min in RP-HPLC chromatograms are U1-viperitoxin-Dr1b and U1-viperitoxin-Dr1a, respectively.
Fig 4.
Intact protein analysis chromatogram of MALDI-TOF: the intact mass of U1-viperitoxin-Dr1b is 13.564 kDa.
Fig 5.
In-vitro myotoxicity of whole venom (Venom) of Sri Lankan Russell’s viper, U1-viperitoxin-Dr1a (Toxin A) and U1-viperitoxin-Dr1b (Toxin B) compared to controls: (a), Concentration-dependent inhibition of direct twitches in chick biventer nerve-muscle preparation by whole venom. (* the 80μg/ml venom group is significantly different from 50 μg/ml group as well as the control group at 170 min; p<0.05: One-way ANOVA followed by Bonferroni’s post-hoc test, n = 4.). (b), Effect of 80μg/ml venom alone and in the presence of antivenom on the response of the muscle to 40mM KCl. (* significantly different compared to the response to KCl obtained prior to the addition of venom; p<0.05: paired t-test) (c), Concentration-dependent inhibition of the direct twitches in chick biventer nerve-muscle preparation by U1-viperitoxin-Dr1b. *(twitch height significantly lower than the control group: p<0.05, one-way ANOVA followed by Bonferroni’s post-hoc test; n = 3–5); (d), Concentration-dependent inhibition of the direct twitches in chick biventer nerve-muscle preparation by U1-viperitoxin-Dr1a. (* twitch height significantly lower than the control group: p<0.05, one-way ANOVA followed by Bonferroni’s post-hoc test; n = 3–5); (e), Effect of 3 μM U1-viperitoxin-Dr1a (B) and U1-viperitoxin-Dr1b (M) towards the response of the muscle for 40 mMKCl. (* the KCl response of the venom with and without antivenom, at 170min were significantly reduced compared to the initial responses; p<0.05: paired t-test); (f), Effect of 50μg/ml whole venom versus U1-viperitoxin-Dr1a, U1-viperitoxin-Dr1b, venom without U1-viperitoxin-Dr1a, venom without U1-viperitoxin-Dr1b, both toxins together, venom without both toxins of a same amount of venom in 5ml organ bath towards the response of the muscle for 40 mMKCl. * (* the KCl response is significantly lower compared to the control group at 170 min; p<0.05: one-way ANOVA followed by Bonferroni’s post-hoc test; n = 3–5).
Fig 6.
Alignment of U1-viperitoxin-Dr1b amino acid sequence with U1-viperitoxin-Dr1a.
Sequences were obtained from UniProt database and are presented with unique identification numbers and entry names. In residues marked as ‘*’ are single fully conserved residues. At the positions highlighted in yellow, ‘:’ and ‘.’ denote positions with conservation between groups of strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix) and, conservation between groups of weakly similar properties (scoring = < 0.5 in the Gonnet PAM 250 matrix) respectively.