Wilson Fernandes

Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest.

Bothrops jararaca is a slender and semi-arboreal medically relevant pit viper species endemic to tropical and subtropical forests in southern Brazil, Paraguay, and northern Argentina (Misiones). Within its geographic range, it is often abundant and is an important cause of snakebite. Although no subspecies are currently recognized, geographic analyses have revealed the existence of two well-supported B. jararaca clades that diverged during the Pliocene ~3.8Mya and currently display a southeastern (SE) and a southern (S) Atlantic rainforest (Mata Atlântica) distribution. The spectrum, geographic variability, and ontogenetic changes of the venom proteomes of snakes from these two B. jararaca phylogroups were investigated applying a combined venom gland transcriptomic and venomic analysis. Comparisons of the venom proteomes and transcriptomes of B. jararaca from the SE and S geographic regions revealed notable interpopulational variability that may be due to the different levels of population-specific transcriptional regulation, including, in the case of the southern population, a marked ontogenetic venom compositional change involving the upregulation of the myotoxic PLA2 homolog, bothropstoxin-I. This population-specific marker can be used to estimate the proportion of venom from the southern population present in the B. jararaca venom pool used for the Brazilian soro antibotrópico (SAB) antivenom production. On the other hand, the southeastern population-specific D49-PLA2 molecules, BinTX-I and BinTX-II, lend support to the notion that the mainland ancestor of Bothrops insularis was originated within the same population that gave rise to the current SE B. jararaca phylogroup, and that this insular species endemic to Queimada Grande Island (Brazil) expresses a pedomorphic venom phenotype. Mirroring their compositional divergence, the two geographic B. jararaca venom pools showed distinct bioactivity profiles. However, the SAB antivenom manufactured in Vital Brazil Institute neutralized the lethal effect of both venoms to a similar extent. In addition, immobilized SAB antivenom immunocaptured most of the venom components of the venoms of both B. jararaca populations, but did not show immunoreactivity against vasoactive peptides. The Costa Rican bothropic-crotalic-lachesic (BCL) antivenom showed the same lack of reactivity against vasoactive peptides but, in addition, was less efficient immunocapturing PI- and PIII-SVMPs from the SE venom, and bothropstoxin-I, a CRISP molecule, and a D49-PLA2 from the venom of the southern B. jararaca phylogroup. The remarkable paraspecificity exhibited by the Brazilian and the Costa Rican antivenoms indicates large immunoreactive epitope conservation across the natural history of Bothrops, a genus that has its roots in the middle Miocene. This article is part of a Special Issue entitled: Omics Evolutionary Ecolog.

Semen Collection and Evaluation in Free-Ranging Brazilian Rattlesnakes (Crotalus durissus terrificus)

Two hundred-ninety species of reptiles are estimated to need urgent action for conservation, with at least 113 threatened species worldwide. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species includes 80 species of snakes, with six native Brazilian species, a number likely to be an underestimation. Some authors believe that assisted reproduction would be an important tool to improve reproduction in captivity of some reptiles. An efficient technique for semen collection and evaluation is an important step in development of protocols for cryopreservation of semen or artificial insemination in snakes, contributing to the conservation of endangered species. Although these techniques are important, some basic semen parameters are described for four of the approximately 2,900 snake species in the world. The Brazilian rattlesnake (Crotalus durissus terrificus) was chosen as a model for semen collection in snakes because it is found quite often in Sao Paulo State. Semen was collected once from each animal by the same investigator during the mating season of this species in Brazil. After antiseptic cleansing of the skin around the cloaca, the snakes were injected subcutaneously with a dose of 15 mg/kg of 1% solution of lidocaine around the cloaca. Semen was collected with ventral massages after cloacal relaxation and directly from genital papilla inside the cloaca. A total of 28 ejaculates from 39 animals were obtained, representing collection efficiency of 71.80%. Semen volume and concentration in Brazilian rattlesnakes ranged from 3-70 microl and from 0.94-2.23 x 10(9) spermatozoa/ml, respectively. Zoo Biol 0:1-6, 2007. (c) 2007 Wiley-Liss, Inc.

Influenza em animais heterotérmicos

The objective was to study Orthomyxovirus in heterothermic animals. Blood samples from snakes (genus Bothrops and Crotalus) and from toads and frogs (genus Bufo and Rana) were collected to evaluate the red cell receptors and antibodies specific to influenza virus by the hemagglutination and hemagglutination inhibition tests, respectively. Both snakes and toads kept in captivity presented receptors in their red cells and antibodies specific to either influenza virus type A (human and equine origin) or influenza type B. The same was observed with recently captured snakes. Concerning the influenza hemagglutination inhibition antibodies protective levels were observed in the reptiles’ serum, against influenza type A and type B. Unlike the toads, 83.3% of the frogs presented mean levels of Ab 40HIU for some influenza strains. It was concluded that heterothermic animals could offer host conditions to the influenza virus and also susceptibility to the infection. Key-words: Orthomyxovirus. Heterothermic animals. Receptors. Specific antibodies. Influenza virus. 1. Laboratorio de Virologia do Instituto Butantan, Sao Paulo, SP. 2. Laboratorio de Genetica do Instituto Butantan, Sao Paulo, SP. 3. Laboratorio de Herpetologia do Instituto Butantan, Sao Paulo, SP. Endereco para correspondencia: Dra. Dalva Assuncao Portari Mancini. Divisao de Desenvolvimento Cientifico/Laboratorio de Virologia/IB. Av. Vital Brasil 1500, 05503-900 Sao Paulo, SP. Tel: 55 11 3726-7222 ramal 2152 e-mail: labvirol@bol.com.br dapmancini@butantan.gov.br. Recebido para publicacao em 8/2/2003 Aceito em 27/2/2004 Para melhor entendimento sobre a funcao do receptor celular no reconhecimento do virus influenza, tem sido desenvolvidas pesquisas relacionadas a especificidade de ligacao desse virus isolado de varias especies animais 10 . Bossart e cols verificaram atraves da microscopia eletronica, os fenomenos conhecidos de adsorcao (atraves da hemaglutinina) e subsequente eluicao (pela neuraminidase) do virus influenza sobre a membrana de eritrocitos de ave e de humano. Essa interacao do virus a celula vermelha e comparado ao que ocorre as celulas hospedeiras. Os virus influenza tipo A mostram habilidade, entre seus subtipos, de ligacao aos receptores atraves dos terminais dos acidos sialicos, contendo oligossacarideos, mas, sao distintos quanto a especificidade de receptor, o que provavelmente os diferencia quanto a virulencia aos hospedeiros . Quanto ao tropismo desse virus, verifica-se que as celulas receptoras virus ARTIGO/ARTICLEO objetivo foi pesquisar Ortomyxovirus em animais heterotermicos. Coletou-se sangue de serpentes dos generos Bothrops e Crotalus e de sapo e ras dos generos Bufo e Rana, para a deteccao dos receptores de hemacias e anticorpos especificos, ao virus influenza, pelos testes de hemaglutinacao e inibicao da hemaglutinacao, respectivamente. Pelo teste de hemaglutinacao, verificou-se que serpentes e sapos em cativeiro apresentaram receptores em suas hemacias para o virus influenza, humano e equino do tipo A e tipo B. O mesmo ocorreu com serpentes recem chegadas. Quanto ao teste de inibicao da hemaglutinacao dos soros dos repteis observou-se titulos protetores de anticorpos aos virus influenza tipo A (origens humana e equina) e tipo B. Com soro de sapo nao se observou reacao de inibicao da hemaglutinacao porem, 83,3% das ras obtiveram medias de 40UIH para algumas cepas. Conclui-se que animais heterotermicos podem oferecer condicoes de hospedeiros aos virus influenza, assim como susceptibilidade a infeccao.

Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes.

Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life.

Examination of biochemical and biological activities of Bothrops jararaca (Serpentes: Viperidae; Wied-Neuwied 1824) snake venom after up to 54 years of storage

ABSTRACT The number of snakes donated to the Brazilian Instituto Butantan has been decreasing in the past 10 years. This circumstance motivated us to compare the properties of five venom pools of Bothrops jararaca snake stored for up to 54 years. Results showed differences among venom pools regarding enzymatic and other biological activities, such as caseinolytic, phospholipase A2, hemorrhagic and coagulant activities, as well as antigenicity. Protein content, reverse‐phase chromatographic profile, and immunorecognition by commercial Bothrops antivenom were comparable for all venom pools, although lethality of the most recent preparations was higher. Since the lowest functional activities did not always correspond to older venoms, differences among venom pools used for antivenom production during the period 1963–2008 may correlate with the different proportions of venoms from different localities used in their generation, rather than to long‐term storage. We conclude that B. jararaca venoms properly stored for long periods of time retain their structural and pharmacological activities, thus representing useful materials for scientific research and antivenom production. Graphical abstract Figure. No Caption available. HighlightsBioactivities of Bothrops jararaca venom pools stored for up to 54 years were analyzed.Protein content, chromatographic and immunorecognition profile were comparable for all venom pools.Lethality of lyophilized venom pools (1997 and 2008) was higher than that of venom pools dried at RT (1963, 1973, 1977‐88).Vintage venom collections represent valuable materials for research purposes and possibly for the production of antivenom.

Viperid venom glands with defective venom production. Morphological study

The venom of viperid snakes is collected monthly at Butantan Institute for research purposes and production of antivenoms. Here we describe histological and ultrastructural changes on Crotalus durissus terrificus and Bothrops sp. venom glands with defective venom production. Secretory tubules commonly showed partial or total obliteration of their lumina by masses of necrotic cells and cellular debris. Secretory cells showed varying degrees of degenerative and/or metaplastic alterations seriously affecting the structures responsible for the synthesis and secretion of venom. The intertubular connective tissue presented fibroblast hyperplasia, inflammatory cells infiltration, vacuolated cells and blood vessels alterations. In two venom glands out of nineteen snakes examined, virus-like particles were found. The alterations observed in most of the glands could have been caused by excessive manual pressure, during venom extraction routine, causing disruption of the secretory tubules and leakage of venom to the intertubular connective tissue.

Alteration of the protein composition of Bothrops jararaca venom and venom gland by isoproterenol treatment

Abstract 1. 1. The protein composition of Bothrops jararaca venom and venom gland was analyzed through SDS-PAGE, after isoproterenol (IPR) treatment. 2. 2. Some proteins (47, 48, 57 and 72 kDa) were detected in the gland homogenate from the control but not from the IPR-treated samples. 3. 3. Three proteins (26.5, 44.5 and 53 kDa) were detected in the venom gland from IPR-treated snakes but not from the venom gland from the control. 4. 4. In the venom samples proteins of 41 and 74 kDa were detected only in the IPR treated samples, while proteins of 17 and 28 kDa were detected only in the control. 5. 5. The biological activity of the venom did not change with IPR treatment.