Characterising venom variation and antivenom effectiveness within the “True” vipers (Viperinae: Viperidae)

Abstract

Venom is a functional adaptation that has evolved independently in almost every major animal lineage for use in defence, predation, and/or competition. The complexity and diversity of components formulating the venom mixture has resulted in a phenomenon whereby the composition of a species’ venom can vary down to the level of the individual. Venom variation produces an undesirable outcome from an anthropogenic perspective as the efficacy of an antivenom may be compromised due to variation in the antigenicity of venoms. This complicates the research efforts that seek to ameliorate the global impact of snakebite. The Viperinae subfamily of vipers contains some of the most medically significant snakes world-wide, often responsible for majority proportions of snakebite in the regions they inhabit. Despite their medical importance, Viperinae is an understudied clade. There is a need to characterise the bioactivity, composition, and variation of venom in these snakes in order to develop a deeper understanding of the complex interactions between snake ecology, evolution, venom, and snakebite envenoming. Accordingly, the aim of this work is threefold: 1. To characterise the venom activity of select viperine genera that are underrepresented in the existing literature, focussing on venom variation; 2. To explore links in the evolutionary relationships between snake ecology and venom biochemistry; and 3. To investigate the effectiveness of antivenoms relative to venom activity and variability. The thesis begins with a broad overview of the evolutionary history, biology, and ecology of the viperines and their venom in Chapter 1. Chapter 2 contains study protocols. Chapter 3, which comprises three sub-chapters, focusses on the genus Daboia. It begins with a characterisation of the proteolytic and haemotoxic properties of venoms from twelve geographical variants of five Daboia species, followed by an assessment of antivenom cross-reactivity, and finishes with a critical comparative analysis of the limitations and advantages offered by a suite of commonly used antivenom assessment protocols. Chapter 4 consists of two sub-chapters focussing on the neglected genera Pseudocerastes and Eristicophis. The first section of the chapter entails a comprehensive analysis of the neurotoxic, haemotoxic, and cytotoxic bioactivities of the venoms, and the second section evaluates antivenom cross-reactivity for the clade. The thesis is concluded in chapter 5.These research aims were achieved by adopting an integrated proteomic, bioactivity, and immunogenic assay approach, and the venoms of Daboia, Pseudocerastes, and Eristicophis were evaluated using all or a combination of the following methods: Venom protein composition was assessed via SDS-PAGE gel analysis. Generalised proteolytic activity, factor X and prothrombin activation, and total thrombin generation by the venoms were assessed via kinetic fluorescence assays. Coagulant activity of venoms on human plasma was quantified using an automated coagulation analyser. Venom-induced kinetics and structure of human, amphibian, and avian plasma clots were assessed via thromboelastography. Activity of venoms on fibrinogen was evaluated by thromboelastography and imaged via fibrinogen gels. Interactions of the venoms with phospholipid, tissue factor, and plasminogen activator were testing using chromogenic assays. Neurotoxicity testing was conducted via chick biventer assays. Cytopathic activity testing was conducted on PaTu-T, NFF, and MM96L cells, quantified via MTT assays, and visualised via light microscopy. Antivenom effectiveness was assessed functionally by using an automated coagulation analyser to measure shifts in coagulation time, as well as immunogenically via western blotting and ELISAs (endpoint titration and chaotropic). The advantages and limitations of a range of antivenom assessment assays were also evaluated.The previously unknown venom activities of Pseudocerastes urarachnoides were revealed to exert strong procoagulant activity, activating both prothrombin and factor X. Most other species tested also possessed strong, procoagulant venoms, with exception of the Afro-Arabian species of Daboia (D. palaestinae and D. deserti) and two species of Pseudocerastes (P. persicus and P. fieldi). Fibrinogenolytic activity across the clade was extremely diverse. Intragenus and intraspecies variation in venom activity was extreme throughout all assays. The variability of these venoms typically had a detrimental impact on the effectiveness of antivenoms, whereby many antivenoms proved inadequate. Their neutralisation of venom activities was inconsistent and ranged from good coverage for some species’ venoms to no coverage for others. These studies present the first comprehensive functional analyses of Pseudocerastes and Eristicophis venoms, representing a key contribution to our knowledge of viper venom composition and activity. Crucially, antivenom effectiveness and cross-reactivity for this clade has been described for the first time. In addition, this work has given greater depth and breadth to the scope of research that exists surrounding the geographic variation in venom composition of Daboia and the impact this has on antivenom effectiveness. This will aid in the treatment of envenomings by thesespecies, and thus positively contributes to the global efforts seeking to address snakebite mortality and morbidity. This series of studies provides a critical data set which informs antivenom manufacturers, clinicians, and evolutionary toxinologists in the field of venom research.