Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization.

BACKGROUND: Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation. The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, in Brazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit the South and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence of intra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was to investigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions of Brazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one (M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic cross-reactivity and the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of protein composition and toxicity revealed a large diversity of venoms from the nine Micrurus species. ELISA and Western blot assays showed a varied capability of the therapeutic antivenom to recognize the diverse species venom components. In vivo and in vitro neutralization assays indicated that the antivenom is not able to fully neutralize the toxic activities of all venoms. CONCLUSION: These results indicate the existence of a large range of both qualitative and quantitative variations in Micrurus venoms, probably reflecting the adaptation of the snakes from this genus to vastly dissimilar habitats. The data also show that the antivenom used for human therapy in Brazil is not fully able to neutralize the main toxic activities present in the venoms from all Micrurus species occurring in the country. It suggests that modifications in the immunization scheme, with the inclusion of other venoms in the antigenic mixture, should occur in order to generate effective therapeutic coral snake antivenom.

Antigenic cross-reactivity and immunogenicity of Bothrops venoms from snakes of the Amazon region.

Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to health services, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata are thought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression of toxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that may guide the improvement of anti-Bothropic therapeutic serum.

Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization

Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation.The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, inBrazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit theSouth and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence ofintra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was toinvestigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions ofBrazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one(M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic crossreactivityand the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms

Antigenic cross-reactivity and immunogenicity of Bothrops venomsfrom snakes of the Amazon region

Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to healthservices, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata arethought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression oftoxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that mayguide the improvement of anti-Bothropic therapeutic serum.

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus.

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.

Interspecific variation in venom composition and toxicity of Braziliansnakes from Bothrops genus

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, andthe standard treatment for snakebites is the antivenom therapy. The anti-bothropic serumproduced by Butantan Institute is prepared by the hyperimmunization of horses witha pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothropsmoojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristicsof the venoms from nineteen snakes of the genus Bothrops, responsible for human accidentsin Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes,are rich sources of serine proteases and metalloproteases and the ability of the Braziliananti-bothropic serum to neutralize the proteolytic activity of these venoms were alsotested. The results obtained here show the existence of a large range of variation in thecomposition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropicserum is not able to fully neutralize the toxic activities of all analysed venoms. Thesesuggest that for the preparation of a fully effective therapeutic anti-bothropic serum, othervenoms should be included in the immunization mixture.

Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus).

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. In this work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus) of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipase A2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporus>B. n. paranaensis>B. n. goyazensis. In BC preparations, B. n. goyazensis venom (50 microg/mL) was significantly (p<0.05) more active than B. n. paranaensis and B. n. diporus venoms, which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms (100 microg/mL) produced blockades of 57.4+/-5%, 30+/-3% and 17.4+/-7% (n=3-6 each), respectively. The three venoms inhibited contractures in response to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a long-lasting, concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responses within 45-100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activity and the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among the venoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship with PLA2 activity varied with the preparation used.

Neuromuscular and phospholipase activities of venoms from three subspeciesof Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus)

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. Inthis work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus)of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipaseA2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporusNB. n. paranaensisNB. n. goyazensis. InBC preparations, B. n. goyazensis venom (50 ìg/mL) was significantly (pb0.05) more active than B. n. paranaensis and B. n. diporus venoms,which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms(100 ìg/mL) produced blockades of 57.4±5%, 30±3% and 17.4±7% (n=3–6 each), respectively. The three venoms inhibited contractures inresponse to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a longlasting,concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responseswithin 45–100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activityand the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among thevenoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship withPLA2 activity varied with the preparation used.

Duvernoy's gland secretion of Philodryas olfersii and Philodryas patagoniensis (Colubridae): neutralization of local and systemic effects by commercial bothropic antivenom (Bothrops genus).

Colubrids involved in human envenomation in Brazil are mainly from the genera Helicops, Oxyrhopus, Thamnodynastes and Philodryas. There is a relatively large number of clinical descriptions involving the Xenodontinae snakes, Philodryas olfersii and Philodryas patagoniensis, in human accidents. The most common manifestations of envenomation are local pain, swelling, erythema and ecchymosis and regional lymphadenopathy with normal coagulation. The aims of this study were to characterize the biochemical and biological properties of P. olfersii and P. patagoniensis venoms, and to investigate their immunological cross-reactivities by using both specific antisera and anti-Bothrops sp serum used for human serum therapy in Brazil, in neutralizing the lethal and hemorrhagic effects of these venoms. We show here that P. olfersii e P. patagoniensis venoms present proteolytic and haemorrhagic activities but are devoid of phospholipase A2 activity. Haemorrhage and lethality induced by P. olfersii and P. patagoniensis are associated with metal-dependent proteinases, since EDTA could block these toxic activities. P. olfersii and P. patagoniensis venoms were immunogenic and the antisera produced were able to recognize several bands in P. olfersii, P. patagoniensis venoms in Bothrops jararaca venom.

Duvernoy’s gland secretion of Philodryas olfersii and Philodryaspatagoniensis (Colubridae): Neutralization of localand systemic effects by commercial bothropicantivenom (Bothrops genus)

Colubrids involved in human envenomation in Brazil are mainly from the genera Helicops, Oxyrhopus, Thamnodynastes and Philodryas. There is a relatively large number of clinical descriptions involving the Xenodontinae snakes, Philodryas olfersii and Philodryas patagoniensis, in human accidents. The most common manifestations of envenomation are local pain, swelling, erythema and ecchymosis and regional lymphadenopathy with normal coagulation. The aims of this study were to characterize the biochemical and biological properties of P. olfersii and P. patagoniensis venoms, and to investigate their immunological cross-reactivities by using both specific antisera and anti-Bothrops sp serum used for human serum therapy in Brazil, in neutralizing the lethal and hemorrhagic effects of these venoms. We show here that P. olfersii e P. patagoniensis venoms present proteolytic and haemorrhagic activities but are devoid of phospholipase A2 activity. Haemorrhage and lethality induced by P. olfersii and P. patagoniensis are associated with metal-dependent proteinases, since EDTA could block these toxic activities. P. olfersii and P. patagoniensis venoms were immunogenic and the antisera produced were able to recognize several bands in P. olfersii, P. patagoniensis venoms in Bothrops jararaca venom.

Caracterizacion de las actividades biologicas de los venenos ‘amarillo’ y ‘blanco’ de Crotalus durissus ruruima comparados con el veneno de Crotalus durissus terrificus. Poder neutralizante de los antivenenos frente a los venenos de Crotalus durissus ruruima / Characterization of the biological activities of venoms 'yellow' and 'white' from Crotalus durissus ruruima compared with the venom of Crotalus durissus terrificus. Neutralizing power of antivenoms against the venom of Crotalus durissus ruruima

The biological activities of ‘yellow’ and ‘white’ venom of a rattlesnake Crotalus durissus ruruima Hoge, 1965, found in the savanna-like vegetation (cerrado) of northern Brazil (Roraima) and Venezuela have been studied, and compared to the reference Crotalus durissus terrificus venom. The lethal activity of venoms depended on the inoculation route. The most toxic venom was the white one. The venoms of C. d. terrificus and the yellow of C. d. ruruima had similar lethalities. The yellow venom of C. d. ruruima showed a caseinolytic activity three times higher than that obtained with either the venom of C. d. terrificus or the white one of the C. d. ruruima. Hemorrhagic and necrotic activities were found only in the yellow venom. White and yellow venoms from C. d. ruruima showed a similar action on fibrinogen; this thrombin-like action was greater with C. d. terrificus venom. On histopathological sections local and pulmonary hemorrhage was found only with the yellow venom, but myonecrotic activity was observed with both venoms of C. d. ruruima. Among all antivenoms studied, the anti-bothropic-crotalic was the best at neutralizing hemorrhagic and hemolytic activities. These results suggest that antivenom bothropic-crotalic should be used in the treatment of patients with snakebite by C. d. ruruima: besides its neutralization on lethal activity, it also neutralizes the hemorrhagic activity present in some venoms.

A computation program to analyse data obtained in quantal assays

É feita a descriçäo de um programa de computaçäo que permite a análise de dados obtidos na determinaçäo da potência de drogas que produzam efeitos do tipo tudo-ou nada, quando administradas a animais experimentais. O programa foi aplicado: a) para determinar as potências das preparaçöes de veneno botrópico e crotálico, as quais foram enviadas ao Ministério da Saúde como padröes para determinaçäo de potências de soros anti-peçonhentos neste país; b) para verificar se o dessecamento espontâneo de veneno botrópico a temperatura ambiente produz um decréscimo de sua potência letal; c) para determinar a potência letal do veneno de Bothrops erythromelas; e d) para comparar a sensibilidade ao veneno botrópico de duas linhagens de camundongos geneticamente selecionados