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. 2016 Dec 2;10(12):e0005172.
doi: 10.1371/journal.pntd.0005172. eCollection 2016 Dec.

Clinical and Pharmacological Investigation of Myotoxicity in Sri Lankan Russell's Viper (Daboia russelii) Envenoming

Affiliations

Clinical and Pharmacological Investigation of Myotoxicity in Sri Lankan Russell's Viper (Daboia russelii) Envenoming

Anjana Silva et al. PLoS Negl Trop Dis. .

Abstract

Background: Sri Lankan Russell's viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell's vipers. This study aimed to investigate evidence of myotoxicity in Russell's viper envenoming, response to antivenom and the toxins responsible for myotoxicity.

Methodology and findings: Clinical features of myotoxicity were assessed in authenticated Russell's viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman's correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 μg/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 μM, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity.

Conclusion: The study shows that myotoxicity in Sri Lankan Russell's viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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.
Fig 2
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
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
Fig 4. Intact protein analysis chromatogram of MALDI-TOF: the intact mass of U1-viperitoxin-Dr1b is 13.564 kDa.
Fig 5
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
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.

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