Comparison of Phylogeny,Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A₂ reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.     

Viper and Cobra Venom Neutralization by Alginate Coated Multicomponent Polyvalent Antivenom Administered by the Oral Route

Background

Snake bite causes greater mortality than most of the other neglected tropical diseases. Snake antivenom, although effective in minimizing mortality in developed countries, is not equally so in developing countries due to its poor availability in remote snake infested areas as, and when, required. An alternative approach in this direction could be taken by making orally deliverable polyvalent antivenom formulation, preferably under a globally integrated strategy, for using it as a first aid during transit time from remote trauma sites to hospitals.

Methodology/Principal Findings

To address this problem, multiple components of polyvalent antivenom were entrapped in alginate. Structural analysis, scanning electron microscopy, entrapment efficiency, loading capacity, swelling study, in vitro pH sensitive release, acid digestion, mucoadhesive property and venom neutralization were studied in in vitro and in vivo models. Results showed that alginate retained its mucoadhesive, acid protective and pH sensitive swelling property after entrapping antivenom. After pH dependent release from alginate beads, antivenom (ASVS) significantly neutralized phospholipaseA₂ activity, hemolysis, lactate dehydrogenase activity and lethality of venom. In ex vivo mice intestinal preparation, ASVS was absorbed significantly through the intestine and it inhibited venom lethality which indicated that all the components of antivenom required for neutralization of venom lethality were retained despite absorption across the intestinal layer. Results from in vivo studies indicated that orally delivered ASVS can significantly neutralize venom effects, depicted by protection against lethality, decreased hemotoxicity and renal toxicity caused by russell viper venom.

Conclusions/Significance

Alginate was effective in entrapping all the structural components of ASVS, which on release and intestinal absorption effectively reconstituted the function of antivenom in neutralizing viper and cobra venom. Further research in this direction can strategize to counter such dilemma in snake bite management by promoting control release and oral antivenom rendered as a first aid.     

利用生物信息学方法和多抗原表位DNA免疫方法研制有效的抗蛇毒血清

传统的抗蛇毒血清是从全毒免疫的马或绵羊的血清中提取,但目前的免疫方法包括对马或绵羊进行的超免也不能达到对大多数临床上重要的毒素都产生免疫应答的目标。目前利物浦大学的Simon. C. Wagstaff等研究人员开发了一种最新的研制抗蛇毒血清的方法—通过鉴定蛇毒金属蛋白酶(SVMPs,SVMPs主要是产生持续性致死性出血,SVMPs很复杂,它具有多种功能型,能作用于多种底物)中有重要临床意义的7个部分,并将它们改造成单链DNA作为免疫原免疫小鼠产生特异抗体。为研发更合理的抗蛇毒血清制备方法提供了依据。     

平颏海蛇毒磷脂酶A2基因的多样性

通过对平颏海蛇毒腺cDNA文库的随机测序和PCR筛选,分离到3种未曾报道的磷脂酶A2（PLA3）cDNA,分别命名为PLA2－8,PLA2－9,PLA2－384。这些cDNA均含有完整的读码框（长度分别为456bp、438bp和438bp）,编码含27个氨基酸残基的信号肽和长度分别为4．8、7．8和8．4。氨基酸序列分析和二级结构预测表明,3种PLA2均属I类磷酯酶A2家族,其中PLA2－8与来自澳大利亚虎蛇Notechis scutatus scutatus的PLA2有81％的同源性;而PLA2－9和PLA2－384则与来自海蛇Enhydrina schistosa的肌毒PLA2有90％的同源性,这不仅预示着他们在功能上可能存在某些相似性,也反映出平颏海蛇PLA2的结构多样性。这组海蛇PLA2同功酶基因的成功克隆为进一步揭示PLA2的结构与功能关系及的分子机制提供了新的信息。     

African Adders: Partial Characterization of Snake Venoms from Three Bitis Species of Medical Importance and Their Neutralization by Experimental Equine Antivenoms

Background

An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms.

Methodology/Principal Findings

The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1–7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.

Conclusion

These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.     

蛇毒的采集与利用

蛇毒是毒蛇头部两侧皮肤下方毒腺的分泌物,在咬啮物体时从毒牙流出,是含多种活性酶的蛋白质。蛇毒虽能伤害人畜,但若能善加利用,可化害为利而成为良药。此外,还可从中提取许多难得的成分作为现代科学研究之用。因而,被誉为“液体黄金”的蛇毒,具有极高的经济价值。现将蛇毒的采集和利用概述如下。     

激素联合抗蛇毒血清环行封闭治疗毒蛇咬伤伤口溃疡

目的探讨激素联合抗蛇毒血清环行封闭治疗毒蛇咬伤伤口溃疡的疗效及实用价值。方法对81例伤口溃疡形成,且周围组织肿胀的毒蛇咬伤患者,在静脉应用抗蛇毒血清的同时,迅速清除及破坏伤口局部蛇毒,伤口常规扩创、负压吸引冲洗,予同种抗蛇毒血清1／2支加甲基强的松龙40mg(或地塞米松10mg)环行封闭,观察治疗后患者自觉症状、伤口愈合程度。结果治疗1～3天后症状明显好转,疼痛减轻,伤口肿胀、溃烂、渗血明显减轻,无1例需植皮或截肢。结论激素联合抗蛇毒血清环行封闭对毒蛇咬伤伤口疗效明显,具有一定的推广价值。     

蛇毒对肿瘤细胞的体外抑制实验

目的 探讨蝮蛇毒及蝮蛇与眼镜蛇混合毒对肿瘤细胞的抗癌活性,方法 应用蝮蛇毒及蝮蛇与眼镜蛇混合毒人源肿瘤细胞进行体外细胞毒试验。结果 蝮蛇毒及蝮蛇与眼镜蛇混合毒对传代细胞株（Ｎｏｖｉｋｏｆｆ及Ｈｅｐ－２）的抑制作用随蛇毒剂理的增加而增强,剂量为５μｇ／ｍｌ时,蝮蛇毒对传代细胞Ｎｏｖｉｋｏｆｆ及Ｈｅｐ－２的抑制率分别为５０．３％和４７．５％,蝮蛇与眼镜蛇混合毒对Ｎｏｖｋｏｆｆ及Ｈｅｐ－２的抑制率分别为     

蛇毒中非成瘾性止痛剂的研究进展

普通止痛药 (去痛片 )长期滥用极易成瘾 ,产生耐受性。因此 ,寻找新的无成瘾性止痛剂具有十分重要的意义。蛇毒替代吗啡用于癌症晚期的镇痛已有报道。目前 ,已有研究毒素的蛇种属主要有Vipera berus,Crotalus adamanteus,C.durissusterrificus,Bothrops alternatus,B.cotiara,B.jararacussu和 N aja naja。止痛有效的产品已从眼镜蛇毒 (Naja naja)中分离得到 ,由于它具有更高的镇痛活性 ,且无成瘾性 ,有可能成为吗啡的重要替代品。从 V.aspis和 V.ammodytes中分离得到的戒毒素在关节炎和风湿症的治疗中也取得了满意效果。另外眼镜蛇毒素…     

Antibodies against Venom of the Snake Deinagkistrodon acutus

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10⁸ and 5.28 × 10⁸ transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.     

Two Reference-Quality Sea Snake Genomes Reveal Their Divergent Evolution of Adaptive Traits and Venom Systems

True sea snakes (Hydrophiini) are among the last and most successful clades of vertebrates that show secondary marine adaptation, exhibiting diverse phenotypic traits and lethal venom systems. To better understand their evolution, we generated the first chromosome-level genomes of two representative Hydrophiini snakes, Hydrophis cyanocinctus and H. curtus. Through comparative genomics we identified a great expansion of the underwater olfaction-related V2R gene family, consisting of more than 1,000 copies in both snakes. A series of chromosome rearrangements and genomic structural variations were recognized, including large inversions longer than 30 megabase (Mb) on sex chromosomes which potentially affect key functional genes associated with differentiated phenotypes between the two species. By integrating multiomics we found a significant loss of the major weapon for elapid predation, three-finger toxin genes, which displayed a dosage effect in H. curtus. These genetic changes may imply mechanisms that drove the divergent evolution of adaptive traits including prey preferences between the two closely related snakes. Our reference-quality sea snake genomes also enrich the repositories for addressing important issues on the evolution of marine tetrapods, and provide a resource for discovering marine-derived biological products.     

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

Genetic Diversity within Cryptosporidium parvum and Related Cryptosporidium Species

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.     

Diversity and Species Relationship in Pearl Millet (Pennisetum typhoides) and Related Species

Thirteen accessions of pearl millet (Pennisetum typhoides (L) Leeke) collected from different states of India and eight wild species of the genus Pennisetum across the world were analyzed for genetic diversity using AFLP markers. A combined analysis of eight primer combinations showed 35% polymorphism among P. typhoides accessions while analysis with five primer combinations showed 99% polymorphism among the wild species. The dendrogram constructed for the P. typhoides accessions based on the UPGMA method revealed two major clusters with samples from Gujarat forming a separate cluster from the rest of the samples. Principal component analysis of the same data set revealed similar results with the first principal component accounting for 65% of the total variation. The percentage of rare and common alleles contributing to the diversity in the sample was analyzed using the Shannon Weiner diversity index. The SW index revealed that the samples from Gujarat contributed significantly to the overall diversity among the accessions. Among accessions of each geographical region, considerable variation was revealed by SW index with samples from Tamil Nadu being most polymorphic. The genetic diversity in the accessions could be utilized for future breeding work. The dendrogram constructed for the wild species revealed the extent of genetic diversity among them. Analysis with one primer combination showed P. typhoides being closer to P. mollissimum than to the other analyzed species.