Genomic, transcriptomic, and epigenomic analysis of a medicinal snake, Bungarus multicinctus, to provides insights into the origin of Elapidae neurotoxins

The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.

Animais peçonhentos e sua trajetória secular para as ações de saúde: do histórico à aplicação / Venomous animals and their secular trajectory for health actions: from history to application / Los animales venenosos y su trayectoria secular para las acciones de salud: de la historia a la aplicación

O livro Os animais peçonhentos na saúde pública expõe um vasto histórico de acidentes envolvendo problemas de saúde provocados por animais peçonhentos. Os autores resgatam trabalhos e vivências, descritos por Louis Pasteur e Vital Brazil, fundamentais para o desenvolvimento de processos da ciência translacional como compreendida hoje, no século XXI. A obra é marcada pela defesa da necessidade de políticas públicas coerentes que sejam capazes de promover ações de capacitação, prevenção, diagnóstico, tratamento e reabilitação dos pacientes.

The book Os animais peçonhentos na saúde pública presents a vast history of accidents involving health problems caused by venomous animals. The authors rescue works and experiences, described by Louis Pasteur and Vital Brazil, fundamental for the development of translational science processes as understood today, in the 21st century. The work is marked by the defense of the need for coherent public policies capable of promoting training, prevention, diagnosis, treatment and rehabilitation of patients.

El libro Os animais peçonhentos na saúde pública expone una vasta historia de accidentes que involucran problemas de salud causados por animals venenosos. Los autores rescatan obras y experiencias, descritas por Louis Pasteur y Vital Brazil, fundamentales para el desarrollo de los procesos de ciencia translacional tal como se entienden hoy, en el siglo XXI. El libro está marcado por la defensa de la necesidad de políticas públicas coherentes capaces de promover la formación, la prevención, el diagnóstico, el tratamiento y la rehabilitación de los pacientes.

Landscape of research, production, and regulation in venoms and antivenoms: a bibliometric analysis / Panorama de la investigación, la producción y la regulación de venenos y antivenenos: un análisis bibliométrico / Panorama de pesquisa, produção e regulação de venenos e antivenenos: análise bibliométrica

ABSTRACT Objectives. To assess the productivity and visibility in research, clinical studies, treatment, use and production of antivenoms against poisonous snakes, scorpions and spiders. Methods. Bibliometric analysis of research and other activities. Articles on venoms and antivenoms published between 2000 and 2020 were retrieved from the Scopus database. The records were analyzed by bibliometric indicators including number of documents per year, journals, authors, and citation frequency. VOSviewer® v.1.6.13 was used to construct bibliometric networks for country co-authorships and co-occurrence of terms. Results. Australia, Brazil, Costa Rica and India were among the six top countries with most documents and were selected for more detailed analysis. Costa Rica was the country with the largest percentage of its publications dedicated to antivenom production and venomics. Only a few papers dealt with the issues of quality, safety, and efficacy of antivenoms or the role of the national regulatory authorities. The use of VOSviewer® allowed visualization through joint publications of networking between countries. Visualization by co-occurrence of terms showed differences in the research carried out. Conclusions. Working in a collaborative and coordinated manner these four countries could have a major impact on envenoming globally. Attention should be given not only to antivenom production but also to strengthening regulatory oversight of antivenom products.

RESUMEN Objetivos. Evaluar la productividad y la visibilidad en la investigación, los estudios clínicos, el tratamiento, el uso y la producción de antivenenos contra las picaduras de serpientes, arañas y escorpiones venenosos. Métodos. Análisis bibliométrico de la investigación y de las otras actividades. Se tomaron los artículos sobre venenos y antivenenos publicados entre el 2000 y el 2020 en la base de datos de Scopus. Estos documentos se analizaron mediante indicadores bibliométricos como el número de documentos por año, revistas, autores o frecuencia en las citas. Se utilizó VOSviewer® v.1.6.13 para crear una red bibliométrica para coautorías de países y coapariciones de términos. Resultados. Australia, Brasil, Costa Rica e India estaban entre los seis primeros países con más documentos y se seleccionaron para un análisis más detallado. Costa Rica fue el país con el mayor porcentaje de sus publicaciones dedicadas a la producción de antivenenos y la venómica. Solo unos pocos artículos trataban los temas de la calidad, la seguridad y la eficacia de los antivenenos, o la función de las autoridades regulatorias nacionales. Gracias a VOSviewer® pudimos visualizar las publicaciones conjuntas de las colaboraciones entre países. La visualización por la coaparición de términos arrojó diferencias en la investigación realizada. Conclusiones. Si estos cuatro países trabajasen de forma colaborativa y coordinada, podrían tener una repercusión mayor en los envenenamientos por picaduras en el mundo. El foco no debe ponerse solo en la producción de antivenenos, sino también en fortalecer la supervisión regulatoria de estos productos.

RESUMO Objetivos. Avaliar a produtividade e visibilidade em pesquisa, estudos clínicos, tratamento, uso e produção de antivenenos contra peçonhas de serpentes, escorpiões e aranhas. Métodos. Análise bibliométrica de pesquisas e outras atividades. Artigos sobre venenos e antivenenos publicados entre 2000 e 2020 foram obtidos da base de dados Scopus. O conteúdo foi analisado segundo indicadores bibliométricos, como número de artigos por ano, periódicos, autores e frequência de citação. Utilizou-se o software VOSviewer® v.1.6.13 para construir redes bibliométricas de coautoria de países e co-ocorrência de termos. Resultados. Austrália, Brasil, Costa Rica e Índia figuraram entre os seis principais países com o maior número de artigos e, assim, foram selecionados para uma análise mais aprofundada. A Costa Rica teve a maior porcentagem de publicações dedicadas à produção de antivenenos e pesquisa em venômica. Apenas um pequeno número de artigos tratou de questões relacionadas à qualidade, segurança e eficácia dos antivenenos ou ao papel das autoridades nacionais reguladoras. O software VOSviewer® permitiu visualizar, através das publicações conjuntas, as redes formadas entre diferentes países. A visualização por co-ocorrência de termos revelou diferenças nas pesquisas realizadas. Conclusões. Trabalhando de forma colaborativa e coordenada, esses quatro países tiveram uma influência importante em nível mundial no campo de acidentes por animais peçonhentos. Deve-se atentar não apenas à produção de antivenenos, mas também ao fortalecimento da fiscalização regulatória destes.

Envenenamiento ofídico en Costa Rica: logros y tareas pendientes / Snakebite envenoming in Costa Rica: achievements and pending tasks

Resumen Objetivo: Efectuar un análisis de los logros en Costa Rica en cuanto al problema de los envenenamientos por mordeduras de serpientes, y señalar tareas pendientes para reducir aún más el impacto de esta patología. Metodología: Se efectuó una revisión de bibliografía relacionada con el estudio del envenenamiento ofídico en Costa Rica y con los avances efectuados en el tema, en el país. Paralelamente, se identificaron aspectos que requieren atención en el manejo de esta enfermedad tropical desatendida. Conclusiones: Desde las primeras décadas del siglo XX, se han realizado avances significativos en la comprensión y manejo del problema de los envenenamientos por mordedura de serpiente en Costa Rica. Se ha trabajado desde una visión integral que incluye: investigación científico-tecnológica, producción y distribución de antivenenos, esfuerzos en prevención, capacitación de las personas profesionales de la salud en el diagnóstico y tratamiento, y docencia de grado y posgrado en el tema. El país ha asumido un papel de liderazgo a nivel internacional, tanto en el plano académico como en la provisión de antivenenos a muchos países. No obstante, aún quedan aspectos del problema que requieren nuevos esfuerzos en nuestro medio, sobre todo en lo referente a la atención de las consecuencias biomédicas, psicológicas, sociales y económicas que sufren las personas afectadas.

Abstract Aim: To analyze the achievements made in Costa Rica in confronting the problem of snakebite envenomings, and to identify pending tasks to further reduce the impact of this pathology. Methods: A review of the literature on snakebite envenomings in Costa Rica was carried out, identifying the main achievements reached in the country. In parallel, issues that require renewed attention in the management of this neglected tropical disease were identified. Conclusions: Since the first decades of the 20th century, significant advances have been made in Costa Rica for understanding and confronting the problem of snakebite envenomings. An integrative perspective has been implemented, which includes scientific and technological research, production and distribution of antivenoms, prevention campaigns, training of health professionals in the diagnosis and treatment of envenomings, and teaching at graduate and undergraduate levels. Costa Rica has had a leading international role in this topic at the academic level, and by providing antivenoms to many countries. Nevertheless, there are issues that require further efforts, especially regarding the attention to the biomedical, psychological, social and economic consequences suffered by people affected by snakebites.

Toxinology in Colombia: contributions of ophidism/scorpionism program and other research groups / Toxinología en Colombia: contribuciones del programa de ofidismo/escorpionismo y de otros grupos de investigación

Background: Toxinology is a sub-field of toxicology dedicated to studying toxins produced by animals, plants and, microorganisms. In Colombia, during the last thirty years, this area has been mainly investigated by Ophidism/Scorpionism Program of Universidad de Antioquia. However, some other research groups have also contributed to our knowledge of venoms and toxins, as well as their related effects and treatments. Objective: to highlight the most significant findings in toxinology made by the Ophidism/Scorpionism Program and other research groups in Colombia. Methods: 119 papers dealing with the history of ophidiology and toxinology in Colombia were collected and analyzed. Results: some useful terms are described to understand toxinology and its scope. Also, a brief history of ophidiology is presented, spanning from the discovery of America until present-day findings. Finally, an overall description of several results related to toxin isolation, characterization, antivenoms, clinical trials, description of new species, proteomic and transcriptomic, among others. The nineteens were characterized by the study of snakebites, their clinic manifestations, and the use of antivenoms. In addition, the ethnopharmacological studies of medicinal plants used in snakebite treatments began to be explored. The 2000s included the newly ethnopharmacology, toxin isolation, clinical trials, inhibitor studies, scorpion venom characterization, and scorpion stings features. Finally, from 2010 until today, proteomic and transcriptomic gave the most important findings. Conclusions: Toxinology works in Colombia have contributed to our knowledge about endemic species, clinical manifestations of snakebite and scorpion stings, and the development of new therapeutic agents. However, we invite Colciencias and other funding agencies to assign more resources to support a higher number of researchers in this field, since snakebite is considered a neglected tropical disease by the World Health Organization, which needs more attention from governments and scholars. Finally, the venoms of some species and their possible mode of action are still unknown to us. Besides, given the complexity of venoms, we are not yet aware of the potential use of toxins in current biomedicine. Thus, studies in toxinology must continue.

Antecedentes: La Toxinología es el campo de la Toxicología que estudia las toxinas producidas por animales, plantas y microorganismos. En Colombia, durante los últimos treinta años, los estudios realizados en esta área han sido desarrollados principalmente por el Programa de Ofidismo/Escorpionismo de la Universidad de Antioquia. Sin embargo, otros grupos de investigación también han contribuido en el conocimiento de venenos, toxinas, efectos y tratamientos. Objetivo: Destacar los hallazgos más relevantes en toxinología realizados por el Programa de Ofidismo Escorpionismo y otros grupos de investigación en Colombia. Métodos: Se recopilaron 119 artículos referentes a la historia de la ofidiología y la toxinología en Colombia. Resultados: Se describieron algunos términos útiles para el entendimiento de la toxinología y sus alcances. Se construyó una breve historia de la ofidiología que inicia con el descubrimiento de América y finaliza con hallazgos recientes. Se realizó una amplia descripción de varios resultados relacionados con el aislamiento y caracterización de toxinas, antivenenos, ensayos clínicos, descripciones de nuevas especies, proteómica y transcriptómica, entre otras. Así, la década de los noventa se caracterizó por el estudio de las mordeduras de serpientes, sus manifestaciones clínicas, el uso de antivenenos y la exploración de la etnofarmacología asociada a las mordeduras de serpiente. La década del 2000 incluyó nuevamente etnofarmacología, el aislamiento de toxinas, ensayos clínicos, estudios sobre inhibidores de toxinas, caracterización de venenos y picaduras de escorpión. Finalmente, desde 2010 hasta hoy, la proteómica y transcriptómica aportaron los hallazgos más importantes. Conclusiones: Los estudios de Toxinología en Colombia han contribuido al conocimiento de especies endémicas, manifestaciones clínicas de mordeduras de serpientes y picaduras escorpiones, y el desarrollo de nuevos agentes terapéuticos. No obstante, se invita a Colciencias y a otras agencias de financiamiento a apoyar la investigación en este campo, ya que es considerada una enfermedad tropical desatendida por la Organización Mundial de la Salud y necesita mayor atención por parte del gobierno e instituciones académicas. Además, dada la complejidad de los venenos, se desconoce el uso potencial de las toxinas en la biomedicina actual. Así, se deben continuar realizando estudios en toxinología.

Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

Objective To explore whether a DNA immunization approach targeting the major haemorrhage molecule of a prothrombin activator-like metalloproteinase from Echis ocellatus (E. ocellatus) venom could be conceived to inspire antibodies with more prominent specificity and equal adequacy to current conventional antivenoms systems. Methods The isolated DNA EoMP-6 was used as the template for PCR amplification using the EoDC-2-specific forward and reverse primers. A PCR product of approximately 700 bp was obtained and cloned into pSecTag-B expression vector where anti-EoDC-2 antibodies were generated and analysed for their efficacy to neutralise local haemorrhage in vitro and in vivo. Results Our results suggest that the generated anti-EoDC-2 showed a remarkable efficacy by (a) interfering with the interaction of the recombinant disintegrin “EoDC-2” isolated from the E. ocellatus as well as other viper species to the α

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

Current challenges for confronting the public health problem of snakebite envenoming in Central America

Snakebite envenoming is a serious public health problem in Central America, where approximately 5,500 casesoccur every year. Panama has the highest incidence and El Salvador the lowest. The majority, and most severe,cases are inflicted by the pit viper Bothrops asper (family Viperidae), locally known as ‘terciopelo’, ‘barba amarilla’ or‘equis’. About 1% of the bites are caused by coral snakes of the genus Micrurus (family Elapidae). Despite significantand successful efforts in Central America regarding snakebite envenomings in the areas of research, antivenommanufacture and quality control, training of health professionals in the diagnosis and clinical management of bites,and prevention of snakebites, much remains to be done in order to further reduce the impact of this medicalcondition. This essay presents seven challenges for improving the confrontation of snakebite envenoming inCentral America. Overcoming these challenges demands a coordinated partnership of highly diverse stakeholdersthough inter-sectorial and inter-programmatic interventions.

New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India.

The direct estimate of 46,000 snakebite deaths in India in 2005 (1 for every 2 HIV/AIDS deaths), based on verbal autopsies, renders unrealistic the total of only 47,000 snakebite deaths in the whole world in 2010, obtained indirectly as part of the “Global Burden of Disease 2010” study. Persistent underestimation of its true morbidity and mortality has made snakebite the most neglected of all the WHO’s “neglected tropical diseases”, downgrading its public health importance. Strategies to address this neglect should include the improvement of antivenom, the only specific antidote to envenoming. To accommodate increased understanding of geographical intraspecific variation in venom composition and the range of snake species that are medically important in India, the design of antivenoms (choice of venom sources and species coverage) should be reconsidered. Methods of preclinical and clinical testing should be improved. The relatively new science of venomics involves techniques and strategies for assessing the toxin composition of snake venoms directly through proteomics-centred approaches or indirectly via high-throughput venom gland transcriptomics and bioinformatic analysis. Antivenomics is translational venomics: a proteomics-based protocol to quantify the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. These approaches could revolutionize the preclinical assessment of antivenom efficacy, leading to a new generation of antivenoms that are clinically more effective.

Cross-neutralization of the coagulant activity of Crotalus durissus terrificus venom from the northeast of Argentina by bivalent bothropic antivenom

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Ethnobotanic study of Randia aculeata (Rubiaceae) in Jamapa, Veracruz, Mexico, and its anti-snake venom effects on mouse tissue

In Mexico, medicinal plants are widely used. The use of Randia aculeata by healers against snakebites has never been scientifically tested in relation to possible effects on blood parameters and muscle tissue damage. Interviews were carried out in Jamapa, Veracuz, Mexico, with local residents to collect information about the traditional use of Randia aculeata. In this locality, seven pieces of fruit from the plant are mixed in a liter of alcohol, and then administered orally against snakebites. By using histological techniques and a murine model, we explored its cytoprotective properties against the effects of Crotalus simus and Bothrops asper venoms. Possible protections provided by the plant against tissue damage to skeletal and cardiac muscles and against the typical loss of red blood cells were analyzed. Randia aculeata caused an increase in microhematocrit and total hemoglobin, parameters that are often decremented in association with the loss of red blood cells, which is a characteristic effect of animal venom. Randia aculeata was also shown to protect against the lowering of platelet levels caused by Bothrops asper venom. Finally, Randia aculeata produced a partial inhibition of necrosis following administration of snake venom in skeletal and myocardial muscles. The present results provide solid evidence for the traditional use of Randia aculeata against snakebites, as demonstrated by protection against muscular tissue damage and the diminution of red blood cells.

Envenenamientos por mordeduras de serpientes en América Latina y el Caribe: Una visión integral de carácter regional / Snakebite poisoning in Latin America and the Caribbean: An integral view from a regional perspective

Se analiza la situación de los envenenamientos por mordeduras de serpiente en América Latina y el Caribe, los cuales representan un importante problema de Salud Pública en la región. Esta patología afecta, fundamentalmente, a la población rural de nuestros países, y tiene un alto impacto en sectores desatendidos por los programas de salud. Estos envenenamientos son causados, en su gran mayoría, por especies de la familia Viperidae, especialmente del género Bothrops. Existe un conglomerado de laboratorios públicos y privados productores de antivenenos en la región, aunque en algunos casos la producción no satisface las necesidades de algunos países, por lo que los antivenenos deben ser importados de países vecinos. Las investigaciones científicas y tecnológicas efectuadas en América Latina han generado un gran bagaje de conocimiento sobre las serpientes y sus venenos, así como sobre la clínica de los envenenamientos y el perfil de eficacia y seguridad de los antivenenos, a niveles preclínico y clínico. Pese a los indudables logros obtenidos en el manejo de esta enfermedad en la región, se debe redoblar esfuerzos para garantizar:

(a) un mejor conocimiento de los venenos y sus efectos; (b) una visión más realista de la incidencia de estos envenenamientos; (c) un mejoramiento cualitativo y cuantitativo en la producción de antivenenos; (d) un mejor control de calidad de los antivenenos que se importan en algunos países; (e) una más adecuada distribución de los antivenenos en la región, especialmente en zonas rurales de alta incidencia de envenenamientos; (f) una mayor capacitación del personal de salud en el tratamiento de estos envenenamientos, incluyendo el correcto uso de antivenenos; (g) un seguimiento y atención a las personas que han sufrido secuelas como producto de estos accidentes; y (h) programas comunitarios de prevención y atención de esta patología. Estas tareas deben ser enfrentadas con una filosofía de equidad, solidaridad y cooperación en la región, con la participación de múltiples protagonistas a muy diversos niveles.

The public health problem of envenomings induced by snakebites in Latin America and the Caribbean is analyzed in this work. This pathology affects predominantly the rural population and has a high impact on regions where the provision of health services is insufficient. The majority of envenomings are inflicted by species of the genera Bothrops and Crotalus, classified in the family Viperidae. There are several laboratories in the region which manufacture antivenoms for the treatment of these envenomings, although the volume of production in some cases does not fulfill the national demand and, consequently, antivenoms have to be imported. A significant body of knowledge has been gained in the taxonomy of the snakes and the biochemistry, toxicology and immunology of venoms, as well as in the preclinical and clinical performance of antivenoms. Despite significant advances in the control of this neglected tropical disease in Latin America, there are pending tasks in the region, such as:

(a) To improve our knowledge on snakes and their venoms; (b) to assess the actual incidence and mortality of snakebite envenomings; (c) to increase the volume of antivenom produced and, in some cases, to improve the quality of antivenoms; (d) to improve the national quality control laboratories; (e) to develop more effective strategies of distribution of antivenoms, especially to remote rural areas where snakebites are frequent; (f) to foster permanent education programs for the health staff in charge of the treatment of these envenomings; (g) to follow up and provide support to people that suffer physical or psychological sequelae as a consequence of these envenomings; and (h) to strengthen community programs aimed at improving the prevention and adequate management of snakebites. This conglomerate of tasks should be approached with a philosophy of solidarity, integration and cooperation in the region, with the involvement of multiple actors and institutions.

What is the optimum concentration of m-cresol in antivenoms?

Antivenoms against snake and scorpion envenomations are usually equine in nature and composed mostly of F(ab')2; additionally, phenol and m-cresol are mainly employed for their preservation. Although there is no study on this subject, m-cresol is utilized by most manufacturers in a concentration that ranges from 0.15 to 0.35 g percent. Decreasing the concentration of m-cresol to its minimal effective level may protect victims from its toxic effects and keep the antivenom stable during its shelf life without forming any aggregates. In the present work, different concentrations of m-cresol, ranging from 0.1 to 0.35 g percent, were used with some selected batches of snake and scorpion antivenoms. A low concentration of 0.15 g percent showed an acceptable preserving result that complies perfectly with antimicrobial specifications stated by the British Pharmacopoeia. Tested antivenoms (in 12 batches), when kept in a cold room for 39 months (more than their shelf life), retained their physical, chemical and microbiological activities according to the specifications of pharmacopeias. The present data demonstrated that reduction of m-cresol concentration to 0.15 g percent in case of equine F(ab')2 antivenoms will improve safety of such preparations and preserve their stability during their shelf life.(AU)