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. 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. 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.
Snake bite is a significant public health issue in the tropics [1]. Coagulopathy, neuromuscular paralysis, acute kidney injury and local effects are the most important clinical syndromes of snake envenoming [2]. Russell’s viper bites cause a large number of envenomings across Asia, and are more medically important than any other snake in the region [3,4]. Both species of Russell’s vipers, i. e. D. russelii (found in Sri Lanka, India, Pakistan, Nepal, Bangladesh) and D. siamensis (found in some parts of southeast and east Asia such as Cambodia, Myanmar, Thailand, Taiwan, South China and, some parts of Indonesia including East Java), commonly cause venom-induced consumption coagulopathy, acute kidney injury and mild local effects throughout their distribution [3,5]. However, there is geographical variation in the clinical effects of Russell’s viper envenoming with neuromuscular paralysis and rhabdomyolysis only reported from Sri Lanka and South India [3,6–9]. In a recent clinical and neurophysiological investigation of neuromuscular paralysis in Sri Lankan Russell’s viper envenoming, we showed that the paralysis is mild, non-life threatening with no long term effects [6]. Clinical evidence of myotoxicity, including local and generalized myalgia, muscle tenderness, and dark red or black coloured urine suggestive of myoglobinuria, has been reported in cases of Russell’s viper envenoming in Sri Lanka [7–10]. Elevation of plasma and urinary myoglobin concentrations were reported in 19 Russell’s viper bite patients from Sri Lanka further suggesting the existence of myotoxicity in Sri Lankan Russell’s viper patients [10]. A recent study of Sri Lankan Russell’s viper venom injected into mice reported an elevation in creatine kinase, also suggesting that the venom is myotoxic in-vivo [11]. Myotoxicity is an important effect of snake envenoming and can manifest locally and systemically [12]. Local muscle necrosis is a component of the local necrotic effects [12,13]. Systemic myotoxicity has been reported following envenoming by some vipers [14,15], sea snakes [16–18], Australasian elapids [19,20] and some Asian kraits [21,22]. Systemic myotoxicity ranges in severity. Loss of functioning muscle cells due to myotoxicity can aggravate co-existing weakness due to neuromuscular block caused by neurotoxins. More importantly rhabdomyolysis can cause secondary acute kidney injury [23,24] and itself can result in life-threatening hyperkalaemia due to extensive muscle cell damage [15]. Although many cytotoxic components in snake venoms may contribute to the development of myotoxicity, the most important snake venom myotoxins are phospholipase A2 (PLA2) toxins [12,25]. Three types of PLA2 myotoxins, commonly referred as Asp49, Ser49 and Lys49 PLA2, have been characterised in viperid venoms. Despite structural similarity, the latter two types lack catalytic activity, and are referred to as ‘PLA2 like’ peptides [26–28]. It is important to note that the enzymatic activity and the myotoxic activity of PLA2 myotoxins are independent [29]. Myotoxic PLA2s cause muscle damage primarily by destruction of the sarcolemma [12,30]. Several PLA2 toxins have been isolated from Sri Lankan Russell’s viper venom and biochemically characterised, [31,32] Some of these PLA2 toxins contain a unique Serine residue at the N-terminus (S-type), while others have an Asparagine residue at the N-terminus (N-type) [32]. We have recently shown that the pre-synaptic neurotoxicity of the Sri Lankan Russell’s viper venom is primarily due to one of the S-type PLA2 toxins, which we named U1-viperitoxin-Dr1a [33]. There are several gaps in our understanding of the myotoxicity associated with Sri Lankan Russell’s viper envenoming. It is unclear if Sri Lankan Russell’s viper envenoming causes severe myotoxicity and whether any resultant myotoxicity can be treated with Indian Polyvalent antivenom. Further, the in vitro myotoxicity of Sri Lankan Russell’s viper venom has not been pharmacologically studied. This requires isolation and pharmacological characterisation of the myotoxins from the venom. The present study aims to investigate the clinical severity of myotoxicity from Russell’s viper envenoming and isolate the toxins responsible for this activity.
The plasma CK concentrations of rats injected with Sri Lankan Russell’s viper venom 250 μg/kg (i. m.) were not different tocontrol rats and were within the normal range until 6 h of the venom injection (S1 Fig). Analysis of U1-viperitoxin-Dr1b by MALDI-TOF mass spectrometry indicated a single species with a mass/charge ratio of 13564 Da (Fig 4). A doubly charged ion with a mass/charge ratio of 6779. 8 Da was also observed.
The present study demonstrates that myotoxicity seen in humans following Sri Lankan Russell’s viper envenoming is mild, based on the relatively low frequency of generalized myotoxic features and only a very mild elevation in CK. There were small but statistically significant association between clinical features of myotoxicity and the 24 h CK measurements. The rats injected intramuscularly with Sri Lankan Russell’s viper venom did not show an elevation of CK within the first 6 h. Although Sri Lankan Russell’s viper venom displays concentration-dependent myotoxic effects in-vitro, very high venom concentrations (i. e. >50 μg/ml) are required to completely inhibit direct twitches in the chick biventer nerve-muscle preparation and the response to KCl. Antivenom at recommended concentrations was unable to neutralise the myotoxic properties of the venom in-vitro. The myotoxicity of the whole venom appears to be due to two abundant PLA2 toxins in the venom, U1-viper1toxin-Dr1a and U1-viperitoxin-Dr1b (named herein), which both have in vitro myotoxic activity, but only at very high concentrations (3 μM). Features of systemic myotoxicity such as generalized muscle tenderness and myalgia, and dark coloured urine were observed in less than 15% of the patients. In addition, three of the six patients with dark coloured urine did not have 24 h CK concentrations outside the normal limit, so the dark urine was more likely due to macroscopic haematuria (due to haemorrhage) which occurs with Russell’s viper envenoming. Peak CK concentrations were abnormally high in less than 17% of patients, and even in these cases there were only modest elevations with the median and maximum being 666 and 1066 U/l, respectively. In comparison, the reported median peak CK values following envenoming by mulga snakes and tiger snakes in Australia were 3100 U/l [20] and 4749 U/l [19], with severe myotoxicity being associated with CK values over 100,000 U/L. This means that myotoxicity in Russell’s viper envenoming is uncommon and mild when it occurs compared to other myotoxic snakes. The median 24 h CK concentrations were higher in symptomatic patients demonstrating that the clinical features were consistent with biochemical evidence of mild muscle injury. Anaesthetised rats injected with venom showed no significant increase in serum CK concentrations compared to saline injected rats, even 6 h after the venom injection. Assuming that the blood volume of a rat with a body weight of 330g is approximately 25 ml, the maximum serum venom concentration that this venom dose (i. e. 250 μg/kg), with 100% systemic absorbance, could give rise to is 3300 ng/ml. This venom concentration is greater than the maximum venom concentration observed in the Russell’s viper envenomed patients in this study, hence the venom dose used for the envenoming is clinically relevant. The absence of a significant CK rise in the envenomed rats during the 6 h observation period may be due to a combined effect of the weak myotoxicity of the venom and the period of observation being too short to observe any CK elevation. These results are not in agreement with the previously reported high CK concentrations (mean: 16,000 U/l) in mice, 3 h after the intramuscular injection of 5 μg (250 μg/kg dose in 20 g mouse) of Sri Lankan Russell’s viper venom [11], despite similar venom doses in the two studies. The reason for the discrepancy is unclear but such a large early increase in CK concentrations is unusual and the mouse model has not previously been validated. The clinical findings were consistent with the in vitro studies in which two low potency myotoxins were found to be relatively abundant in the venom. Very high venom concentrations up to 80 μg/ml (80 times higher than the maximum peak venom concentration observed in envenomed patients) were required to decrease the direct twitch force by two thirds within 3 h. The low potency of both myotoxins was evident from the fact that high concentrations (3 μM) were required for significant in-vitro myotoxic activity. Venom with these two toxins removed did not cause myotoxicity confirming that these toxins are the only major myotoxins in Sri Lankan Russell’s viper venom. Three previous studies between 1984 and 2000 report much higher rates of myotoxicity, based mainly on myalgia and muscle tenderness. In 1984, Jeyarajah [9] reported myotoxic symptoms in 77% of the cases but no biochemical confirmation of myotoxicity. This study included only severe envenoming with 19/22 with acute kidney injury and 6 deaths. In 1988, Phillips et. al. [10] reported myotoxic symptoms in 32% of the cases, but again this only included more severe envenoming (4/23 died). The mean venom concentration of 375 ng /ml (range: 16. 5–702 ng/ml) [10] was much higher compared to the median peak venom concentration of 25 ng/ml (range: 2. 5–2316 ng/ml) in our study [6]. Phillips et al. reported serum and urine myoglobin, which are difficult to interpret because no studies have correlated these measurements with outcomes. They detected myoglobin in the plasma of all 19 patients tested (range: 100->8000; median: 2745, normal value: <50 ng/ml) and in the urine in 14 of 18 patients (110 to >16 000; median: 4000; normal: <21 ng/ml) [10]. Although these appear to be high, much higher values are reported in Australian mulga snake envenoming, where urinary myogolobin values in three patients were 4129,28200 and 127000 ng/ml[20]. In a larger study of 336 patients in 2000 by Kularatne et al [7] there were 47 patients (14%) with myotoxicity based on generalised muscle tenderness. This is consistent with our study in terms of clinical effects but they did not provide any biochemical confirmation. Our 245 patients are typical of current patients. They had high circulating peak venom concentrations with a median of 25ng/ml, with 24 patients having venom concentrations >1000 ng/ml [6]. Further, nearly all had local envenoming, 68% had coagulopathy (half with bleeding manifestations), and 53% had neuromuscular paralysis [6]. Therefore, the mild myotoxic effects in Sri Lankan Russell’s viper envenoming are most likely due to the weak myotoxicity of the venom. In patients with Australian Mulga snake (Pseudechis australis) envenoming, a delay in antivenom treatment has led to an increase in the severity of the myotoxicity [20]. Since there was no control group of patients who did not receive antivenom in this study, it could be argued that the weak myotoxicity observed in Sri Lankan Russell’s viper envenoming is due to an effect of antivenom. However, there was no association between the delay in antivenom treatment and the number of patients having CK >300 U/l, indicating that the weak myotoxicity observed in the patients is not due to an effect of the antivenom. Indian Polyvalent antivenom was used at concentrations equivalent to the tested venom as recommended by the manufacturer. At these concentrations antivenom failed to prevent the myotoxic effects of Sri Lankan Russell’s viper venom in-vitro. Although this may be due to the low efficacy of the antivenom against the myotoxins [39], testing higher antivenom concentrations was not possible due to the practical limitations of increasing antivenom concentration in the tissue organ bath environment without affecting the osmolarity of the physiological salt solution. It was therefore not possible to determine the efficacy of the Indian polyvalent antivenom to neutralise the myotoxic effects of Sri Lankan Russell’s viper venom, because such a large amount of venom was required to cause myotoxicity requiring very high concentrations of antivenom. Of the several ‘s’ type PLA2 toxins isolated from Sri Lankan Russell’s viper venom, VRV-PL-VIIIa [40] has 100% match with the aligned trypsin digested peptide fragments of U1-viperitoxin-Dr1b. The N-terminal sequence of the first 50 amino acids of the toxin ‘P1’ [31] and the N-terminal sequence of first 21 amino acids of the toxin PLA2 4 [32] are a 100% match for the sequence of U1-viperitoxin-Dr1b. Following the suggested rational nomenclature for toxins [41], and given that the 94% sequence homology of the toxin with U1-viperitoxin-Dr1a[33], we have named the above toxin as U1-viperitoxin-Dr1b (Fig 6). The recent study on the venom proteome of the Sri Lankan Russell’s viper [11] reported that five PLA2 toxins make up a relative abundance of 35% of the whole venom, with VRV-PL-VIIIa (U1-viperitoxin-Dr1b) making 13. 9% (as opposed to 22. 2% in our study) of the whole venom. However, the same study did not match a PLA2 with VRV-PL-V (U1-viperitoxin-Dr1a), which makes 19. 2% of the venom in our analysis as previously published [33]. In conclusion, myotoxicity in Sri Lankan Russell’s viper envenoming is mild and non-life threatening, as evident from the low frequency of generalized myotoxicity and low concentrations of serum creatine kinase in envenomed patients. The whole venom of Sri Lankan Russell’s viper has weak myotoxic properties in-vitro. Two PLA2 toxins, U1-viperitoxin-Dr1a and U1-viperitoxin-Dr1b that make 42% of the whole venom, are the major myotoxins in the venom, but display weak myotoxicity. There is a small possibility that myotoxicity may occur in patients with severe Sri Lankan Russell’s viper envenoming in which antivenom is delayed.
There are many gaps in our knowledge of muscle damage caused by snake venoms. Russell’s vipers are more medically important than any other snake in Asia. Sri Lankan Russell’s viper (Daboia russelii) bites have been reported to cause muscle damage in humans, which is not reported for other Russell’s vipers. The aim of the present study was to investigate the onset, severity and resolution of the muscle damage and to identify the toxins responsible for myotoxicity. For this, we studied muscle damage in 245 patients with confirmed Sri Lankan Russell’s viper bites. Patients reported local muscle pain in 72% of cases and generalised muscle pain in 15%. None had severe muscle damage and the symptoms resolved in 80% of patients within 4 days. Measurement of biomarkers of muscle damage in patient blood was consistent with only mild muscle injury, even in patients with symptoms. Two toxins were isolated from Sri Lankan Russell’s viper venom that had similar myotoxic activity to whole venom in chick muscle preparations. Both toxins were weak myotoxins, consistent with what was seen in patients.
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Clinical and Pharmacological Investigation of Myotoxicity in Sri Lankan Russell’s Viper (Daboia russelii) Envenoming