Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. Significance Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey.

Significance: The toxic composition of snake venom varies between species. Such variation can have major medical implications for the treatment of human snakebite victims. Venom variation is largely attributed to differences in toxin-encoding genes present in the genome or venom gland of snakes. Here, we demonstrate that mechanisms affecting the transcription, translation, and posttranslational modification of toxins also significantly contribute to the diversity of venom protein composition. Venom variation observed between related snake species is therefore the result of a complex interaction between a variety of genetic and postgenomic factors acting on toxin genes. Ultimately, this variation results in significant differences in venom-induced pathology and lethality and can undermine the efficacy of antivenom therapies used to treat human snakebite victims.

Summary: Snake venoms are mixtures of numerous proteinacious components that exert diverse functional activities on a variety of physiological targets. Because the toxic constituents found in venom vary from species to species, snakebite victims can present with a variety of life‐threatening pathologies related to the neurotoxic, cytotoxic and haemotoxic effects of venom. Of the 1·8 million people envenomed by snakes every year, up to 125 000 die, while hundreds of thousands survive only to suffer with life‐changing long‐term morbidity. Consequently, snakebite is one of the world's most severe neglected tropical diseases. Many snake venoms exhibit strong haemotoxic properties by interfering with blood pressure, clotting factors and platelets, and by directly causing haemorrhage. In this review we provide an overview of the functional activities of haemotoxic venom proteins, the pathologies they cause in snakebite victims and how their exquisite selectivity and potency make them amenable for use as therapeutic and diagnostic tools relevant for human medicine.

From offense to defense: Venom in snakes is largely used to subdue and/or kill prey, and most venoms have clear actions that facilitate death or paralysis. In one group of snakes, however, venom has evolved and shifted from predation to protection. Specifically, in three different lineages of “spitting” snakes, venom is used to deter predators. Kazandjian et al. show that similar adaptions have occurred within these lineages that transform cytotoxic components into a mixture that acts on mammalian sensory neurons and causes pain. The authors argue that increased predation on these lineages led to similar shifts in venom function. Science , this issue p. 386

Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa. We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a ‘gold standard’ comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom. None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of ‘test’ antivenom IgGs to bind venom proteins were not substantially different from that of the ‘gold standard’ antivenoms. The least effective antivenoms had the lowest IgG content/vial. Manufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-efficacy testing facilities throughout the continent. Snakebite is one of the most under-researched, under-resourced high morbidty/high mortality NTDs, as reflected by the fact that many of the antivenoms used to treat snakebite victims in sub-Saharan Africa are of uncertain and untested efficacy. This Kenya case study is the first examination of the preclinical efficacy of all available antivenoms to neutralize the venom toxic effects of the most medically important snakes in any region of sub-Saharan Africa. Our results identify serious preclinical efficacy limitations in two of the most commonly used antivenoms, that no single antivenom is effective against all regionally important snakes and that the least effective antivenoms had the lowest IgG concentrations. It is our aim that Ministry of Health medicine-supply regulators can use this data as evidence to demand more detailed efficacy evidence from manufacturers, and to justify the establishment of national/regional preclinical testing facilities. We hope this publication will also alert physicians treating African snakebite victims to check the efficacy of antivenom in their pharmacies. We have carefully qualified the extent and limitation of the results and of our interpretation of the clinical implications thereof.