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Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulation in elapids is largely unexplored. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake,Pseudonaja textilis), in addition to comparative genomics, to identifycis- andtrans- acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridisandC. tigris). Although there is conservation in high-level mechanistic pathways regulating venom production, there are histone methylation, transcription factor, and microRNA regulatory differences between these two snake families. Histone methyltransferases (KMT2A, KMT2C and KMT2D) and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFIcis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target different toxin transcripts. Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin evolution between these two snake families, demonstrating multiple toxin genes and regulatory mechanisms converged to underpin a highly venomous phenotype.

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

Snake venoms are primarily composed of proteins and peptides, and these toxins have developed high selectivity to their biological targets. This makes venoms interesting for exploration into protein evolution and structure–function relationships. A single venom protein superfamily can exhibit a variety of pharmacological effects; these variations in activity originate from differences in functional sites, domains, posttranslational modifications, and the formations of toxin complexes. In this review, we discuss examples of how the major venom protein superfamilies have diversified, as well as how newer technologies in the omics fields, such as genomics, transcriptomics, and proteomics, can be used to characterize both known and unknown toxins.Because toxins are bioactive molecules with a rich diversity of activities, they can be useful as therapeutic and diagnostic agents, and successful examples of toxin applications in these areas are also reviewed. With the current rapid pace of technology, snake venom research and its applications will only continue to expand.

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Nieman-Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode for divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty acid-binding residues of ant NPC2 proteins. We identified four and one mosquito NPC2 proteins as potential sterol- and fatty acid-binding proteins, respectively. From the published data, we analysed the expression of NPC2 genes in various tissues and their differential expression in midgut and salivary gland post-arbovirus infections. NPC2 genes are downregulated rather than upregulated in virus-infected tissues. Interestingly, AAEL012064 is the only gene that is downregulated in both the midgut and salivary gland in all virus infections. In addition, AAEL001650 is downregulated in the salivary gland infected with CHIKV, DENV2 or ZIKV. This gene in the midgut is downregulated infected with DENV1 but upregulated with DENV2. We studied the variation in cis elements in the promoter regions of two groups of closely related NPC2 genes and the expression of relevant transcription factors (TFs). In the midgut infected with DENV1 or DENV2, six TFs (CRE-BP1, AP1, c-Jun, c-Fos, Odd, and NF-kB) appear to play an opposing role in the expression of AAEL006854. Two TFs (RXR-beta/alpha and USF) have a potential role in the downregulation of AAEL009556 in CHIKV-infected midgut and salivary gland. Similarly, two TFs (COUP and Ftz) may have a key role in the downregulation of AAEL009555 and AAEL009556 in DENV2-infected salivary gland.

Arboviruses such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses infect close to half a billion people per year, and are transmitted through Aedes aegypti bites. Infection-induced changes in mosquito salivary glands (SG) influence transmission by inducing immunity, which restricts virus replication, and by altering saliva composition, which influences skin infection. Here, we profiled SG responses to DENV2, ZIKV and CHIKV infections by using high-resolution quantitative proteomics. We identified 218 proteins related to immunity, blood-feeding or cellular machinery. We observed that 58, 27 and 29 proteins were regulated by DENV2, ZIKV and CHIKV infections, respectively. While the regulation patterns were mostly virus-specific, we determined the function of four uncharacterized proteins that were upregulated by all three viruses. We revealed the anti-ZIKV function of gamma-interferon responsive lysosomal thiol-like (GILT-like), the anti-CHIKV function of adenosine deaminase (ADA), the pro-ZIKV function of salivary gland surface protein 1 (SGS1) and the antiviral function against all three viruses of an uncharacterized protein we called salivary gland broad-spectrum antiviral protein (SGBAP). The comprehensive description of SG responses to three global pathogenic viruses and the identification of new restriction factors improves our understanding of the molecular mechanisms influencing transmission.

Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization. Venomous snakes have evolved to produce venom, which is a complex mixture of toxic proteins and peptides, both enzymatic and nonenzymatic in nature. In this current era of high-throughput technologies, venomics projects, which include genome, transcriptome, and proteome analyses of various venomous species, have been conducted to characterize divergent venom phenotypes and the evolution of venom-related genes. Additionally, venomics can also inform about mechanisms of toxin production, storage, and delivery. Venomics can guide antivenom and therapeutic strategies against envenomations and identify new toxin-derived drugs/tools. One potentially promising drug development direction is the use of endogenous inhibitors present in snake venom glands and serum that could be useful for snakebite therapeutics. These inhibitors suppress the activity of venom proteases, enzymatic proteins responsible for the irreversible damage from snakebite. This book chapter will focus on insights from venomous snake adaptations, such as the evolution of venom proteases to generate diverse activities and snake natural resistance to inhibit activity, and how this information can inform and have applications in the treatment of venomous snakebite.

Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. In this review, we highlight several examples, from both published and unpublished work in our lab, demonstrating how a combined venom gland transcriptome and proteome methodology allows for comprehensive characterization of venoms, including those from understudied rear-fanged snake species, and we provide recommendations for using these approaches. When characterizing venoms, peptide mass fingerprinting using databases built predominately from protein sequences originating from model organisms can be disadvantageous, especially when the intention is to document protein diversity. Therefore, the use of species-specific venom gland transcriptomes corrects for the absence of these unique peptide sequences in databases. The integration of transcriptomics and proteomics improves the accuracy of either approach alone for venom profiling.

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. We also describe a transcriptomic response associated to apoptosis, blood-feeding and lipid metabolism. The three viruses differentially regulate components of Toll, Immune deficiency (IMD) and c-Jun N- terminal Kinase (JNK) pathways. However, silencing of the Toll and IMD pathway components showed variable effects on SG infection by each virus. In contrast, regulation of the JNK pathway produced consistent responses in both SGs and midgut. Infection by the three viruses increased with depletion of the activator Kayak and decreased with depletion of the negative regulator Puckered. Virus-induced JNK pathway regulates the complement factor, Thioester containing protein-20 (TEP20), and the apoptosis activator, Dronc, in SGs. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. Upon infection in SGs, TEP20 induces antimicrobial peptides (AMPs), while Dronc is required for apoptosis independently of TEP20. In conclusion, we revealed the broad antiviral function of JNK pathway in SGs and showed that it is mediated by a TEP20 complement and Dronc-induced apoptosis response. These results expand our understanding of the immune arsenal that blocks arbovirus transmission., Author summary Arboviral diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses are responsible for large number of death and debilitation around the world. These viruses are transmitted to humans by the mosquito vector, Aedes aegypti. During the bites, infected salivary glands (SGs) release saliva containing viruses, which initiate human infection. As the tissue where transmitted viruses are produced, SG infection is a key determinant of transmission. To bridge the knowledge gap in vector-virus molecular interactions in SGs, we describe the transcriptome after DENV, ZIKV and CHIKV infection using RNA-sequencing and characterized the immune response in this tissue. Our study reveals the broad antiviral function of c-Jun N-terminal kinase (JNK) pathway against DENV, ZIKV and CHIKV in SGs. We further show that it is mediated by the complement system and apoptosis, identifying the mechanism. Our study adds the JNK pathway to the immune arsenal that can be harnessed to engineer refractory vectors.

Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. The three viruses regulate components of Toll, IMD and JNK pathways. However, silencing of Toll and IMD components showed variable effects on SG infection by each virus. In contrast, regulation of JNK pathway produced consistent responses. Virus infection increased with depletion of component Kayak and decreased with depletion of negative regulator Puckered. Virus-induced JNK pathway regulates complement and apoptosis in SGs via TEP20 and Dronc, respectively. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. We identified and characterized the broad antiviral function of JNK pathway in SGs, expanding the immune arsenal that blocks arbovirus transmission.

The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.

Venomous and hematophagous animals use their venom or saliva for survival, to obtain food, and for self-defense. Venom and saliva from these animals are cocktails of bioactive molecules primarily composed of proteins and peptides. These molecules are called toxins because they cause unwanted consequences on prey. They exhibit unique, diverse, and specific biological activities that perturb normal physiological processes of their prey and host. However, the potential of toxins as inspirations for the development of therapeutic agents or pharmacological tools has also long been recognized. In addition to their small size, the exquisite selectivity and structural stability of toxins make them attractive as starting molecule in the development of therapeutic and diagnostic agents. Drug discovery and development from venomous and hematophagous animals against cardiovascular diseases have been particularly successful. Some of the notable success include antihypertensive (captopril and enalapril) and antiplatelet agents (tirofiban and eptifibatide), as well as anticoagulants (lepirudin and bivalirudin). Highlighted in this review are many venom or saliva-derived cardiovascular-active proteins and peptides of therapeutic interest, including those that are currently in preclinical stages and those that have been approved by FDA and currently in the market. The authors attempt to summarize their structure, function, mechanism of action, and development with respect to cardiovascular diseases.

Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3'RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.

Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3'RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.

The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.

Venoms of snakes of the family Elapidae (cobras, kraits, mambas, and relatives) are predominantly composed of numerous phospholipases A2 (PLA2s) and three-finger toxins (3FTxs), some of which are lethal while others are not significantly toxic. Currently, the only identified prey-specific toxins are several nonconventional 3FTxs, and given the large diversity of 3FTxs within Monocled Cobra (Naja kaouthia) venom, it was hypothesized that several 3FTxs, previously found to be non-toxic or weakly toxic 3FTxs in murine models, could potentially be toxic towards non-murine prey. Additionally, it was hypothesized that ontogenetic dietary shifts will be correlated with observable changes in specific 3FTx isoform abundance. Adult and juvenile N. kaouthia venom composition was investigated using ion-exchange FPLC, 1D and 2D SDS-PAGE, mass spectrometry, and various enzymatic and LD50 assays. Alpha-cobratoxin (α-elapitoxin) was the only significantly toxic (LD50

Venom proteins evolve rapidly, and as a trophic adaptation are excellent models for predator–prey evolutionary studies. The key to a deeper understanding of venom evolution is an integrated approach, combining prey assays with analysis of venom gene expression and venom phenotype. Here, we use such an approach to study venom evolution in the Amazon puffing snake, Spilotes sulphureus , a generalist feeder. We identify two novel three-finger toxins: sulditoxin and sulmotoxin 1. These new toxins are not only two of the most abundant venom proteins, but are also functionally intriguing, displaying distinct prey-specific toxicities. Sulditoxin is highly toxic towards lizard prey, but is non-toxic towards mammalian prey, even at greater than 22-fold higher dosage. By contrast, sulmotoxin 1 exhibits the reverse trend. Furthermore, evolutionary analysis and structural modelling show highest sequence variability in the central loop of these proteins, probably driving taxon-specific toxicity. This is, to our knowledge, the first case in which a bimodal and contrasting pattern of toxicity has been shown for proteins in the venom of a single snake in relation to diet. Our study is an example of how toxin gene neofunctionalization can result in a venom system dominated by one protein superfamily and still exhibit flexibility in prey capture efficacy.

Disintegrins are small non-enzymatic proteins common in the venoms of many viperid snakes. These proteins have received significant attention due to their ability to inhibit platelet aggregation and cell adhesion, making them model compounds in drug development and design investigations. The present study used a combination of molecular and proteomic techniques to screen the venom of the Middle American Rattlesnake (Crotalus simus tzabcan) for novel disintegrins. Six disintegrin isoforms were identified, and the most abundant, named tzabcanin, was further isolated and characterized. Tzabcanin consists of 71 amino acids, has a mass of 7105 Da (by MALDI-TOF mass spectrometry) and contains the canonical RGD binding domain. Tzabcanin was not cytotoxic to MCF-7 cells but showed weak cytotoxicity to Colo-205 cells following a 24 h incubation period. Tzabcanin inhibited cell adhesion of both cell lines to immobilized fibronectin and vitronectin, and cell adhesion to immobilized tzabcanin was inhibited when cells were incubated with a cation chelator (EDTA), indicating that integrin–tzabcanin binding is specific. This study provides a detailed analysis of the purification and characterization of tzabcanin and provides sequence and mass data for the multiple disintegrins present in the venom of C. s. tzabcan.

The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake native to northern and eastern regions of Australia, Papua New Guinea and the Solomon Islands. It was inadvertently introduced onto the island of Guam during the late 1940's to early 1950's, and it has caused massive declines and extirpations of the native bird, lizard, and mammal populations. In the current study, we report the characterization of the venom proteome of an adult and a neonate B. irregularis specimens from Guam by a combination of venom gland transcriptomic and venomic analyses. Venom gland transcriptomic analysis of an adult individual identified toxins belonging to 18 protein families, with three-finger toxin isoforms being the most abundantly expressed transcripts, comprising 94% of all venom protein transcript reads. Transcripts for PIII-metalloproteinases, C-type lectins, cysteine-rich secretory proteins, acetylcholinesterases, natriuretic peptides, ficolins, phospholipase A2 (PLA2) inhibitors, PLA2s, vascular endothelial growth factors, Kunitz-type protease inhibitors, cystatins, phospholipase Bs, cobra venom factors, waprins, SVMP inhibitors, matrix metalloproteinases, and hyaluronidases were also identified, albeit, at very low abundances ranging from 0.05% to 1.7% of the transcriptome. The venom proteomes of neonate and adult B. irregularis were also both overwhelmingly (78 and 84%, respectively) dominated by monomeric and dimeric 3FTxs, followed by moderately abundant (21% (N) and 13% (A)) CRISPs, low abundance (1% (N), 3% (A)) PIII-SVMPs, and very low abundance ( Significance paragraph The Brown Treesnake (Boiga irregularis) has caused extensive ecological and economic damage to the island of Guam where it has become a classic example of the negative impacts of invasive species. In the current study, we report the first combined transcriptomic and proteomic analysis of B. irregularis venom of Guam origin. The transcriptome of an adult snake contained toxin sequences belonging to 18 protein families, with three-finger toxin (3FTx) isoforms being the most abundant and representing 94% of all venom protein transcript reads. Our bottom-up and top-down venomic analyses confirmed that 3FTxs are the major components of B. irregularis venom, and a comparative analysis of neonate and adult venoms demonstrate a clear ontogenetic shift in toxin abundance, likely driven by dietary variation between the two age classes. Second-generation antivenomics and Western blot analysis using purified anti-Brown Treesnake rabbit serum IgGs (anti-BTS IgGs) showed strong immunoreactivity toward B. irregularis venom. Interestingly, our anti-BTS IgGs did not cross-react with 3FTxs found in several other rear-fanged snake venoms, or against 3FTxs in the venom of the elapid Ophiophagus hannah, indicating that epitopes in these 3FTx molecules are quite distinct.

Venom production facilities keep established colonies of captive snakes to obtain venom for research and antiserum production. Due to strict regulations of importation, some of these colonies are formed with only a small number of initial animals and consist of closely related individuals (sometimes siblings). To understand the effect of long-term captivity on the venom composition and its impact on antiserum production, we analyzed 15 long-term captive Naja naja (Pakistan) originating from two separate venom production colonies using liquid chromatography-mass spectrometry and electrophoresis. The chromatogram produced from each individual cobra venom was found to be different. When the protein molecular masses of the peaks were identified, it was found that all the venoms consisted of the same protein composition, but the concentration of the proteins were different. Although three-finger toxins and phospholipase A(2) enzymes are the major toxic components present in these venoms, there was a clear difference in the amounts of each individual isoform. Such variation may affect the ability of antivenoms in neutralizing the toxic components of the wild type venom.

Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides access to cDNA sequences in the absence of living specimens, even from commercial venom sources, to evaluate important regional differences in venom composition and to study snake venom protein evolution., Author Summary This work demonstrates that full-length venom protein messenger RNAs are present in secreted venoms and can be used to acquire full-length protein sequences of toxins from both front-fanged (Elapidae, Viperidae) and rear-fanged (Colubridae) snake venoms, eliminating the need to use venom glands. Full-length transcripts were obtained from venom samples that were fresh, newly lyophilized, old, field desiccated or commercially prepared, representing a significant advance over previous attempts which produced only partial sequence transcripts. Transcripts for all major venom protein families (metalloproteinases, serine proteases, C-type lectins, phospholipases A2 and three-finger toxins) responsible for clinically significant snakebite symptoms were obtained from venoms. These sequences aid in the identification and characterization of venom proteome profiles, allowing for the identification of peptide sequences, specific isoforms, and novel venom proteins. The application of this technique will help to provide venom protein sequences for many snake species, including understudied rear-fanged snakes. Venom protein transcripts offer important insights into potential snakebite envenomation profiles and the molecular evolution of venom protein multigene families. By requiring only venom to obtain venom protein cDNAs, the approach detailed here will provide access to cDNA-based protein sequences from commercial and other venom sources, facilitating study of snake venom protein composition and evolution.