Cassandra Marie Modahl

Numerous orthoflaviviruses transmitted through the bites of different mosquito species infect more than 500 million people annually. Skin infection at the bite site represents a critical and conserved step in transmission and a deeper understanding of this process will promote the design of broad-spectrum interventions. Here, we identify and characterize a transmission-enhancing viral factor in mosquito saliva that is shared across orthoflaviviruses. Saliva from West Nile virus-infected Culex and Zika virus-infected Aedes contains a viral non-coding RNA, subgenomic flaviviral RNA (sfRNA), within lipid vesicles distinct from virions. Higher concentration of sfRNA in infectious saliva positively correlates with infection intensity in human cells and skin explants. Early sfRNA delivery into transmission-relevant skin cell types and human skin explant demonstrate that sfRNA is responsible for the infection enhancement. Co-inoculation of sfRNA in a mouse model of transmission enhanced skin infection and worsened disease severity, supporting the role of salivary sfRNA as a transmission-enhancer. Mechanistically, salivary sfRNA attenuates early interferon response in human skin cells and skin explants by disrupting MDA5 signaling. Our results, derived from two distinct orthoflaviviruses and supported by prior studies, establish salivary sfRNA as a pan-orthoflavivirus transmission-enhancing factor driven by a conserved viral non-coding RNA.

Background: Many animals change traits over the course of their life history. It has been proposed that some of these developmental changes evolve in concert with shifts in ecology, but there are relatively few clear examples, and identifying the underlying molecular changes is challenging. Australian brown snakes ( Pseudonaja spp.), one of Australia's most venomous snakes, may provide such an example, as previous work has shown that these species undergo a dietary shift from reptiles to mammals, accompanied by differences in venom activity. Results: We show that the venom composition of adult brown snakes is distinguished by the collective expression of four procoagulant toxin families, which are absent in juvenile life stages, where venom composition is instead dominated by neurotoxins. The developmental onset of expression across all four procoagulant toxins correlates with a functional shift in venom activity to one that disrupts the blood-clotting system of mammalian prey. We further show that this developmental timing in both toxin expression and procoagulant venom activity coincides with a broader ecological transition in which Australian brown snakes begin to prey on mammals. In contrast, we found that the smallest brown snake species, a lizard specialist throughout its lifetime, only expresses neurotoxins and shows no comparable developmental shift in either procoagulant toxin expression or procoagulant activity. Conclusion: Our findings suggest that a coordinated shift in the expression of four procoagulant toxin gene families transitions the venom from a neurotoxic phenotype in lizard-feeding juveniles to a potent procoagulant phenotype in mammal-feeding adults. These results provide a clear example of how the concerted upregulation of multiple genes can drive a functional shift in a key ecological trait, linking changes in toxin gene expression to venom activity and, ultimately, to prey-capture strategy.

Non-front-fanged snakes are abundant, diverse and represent approximately 70% of extant snakes. However, there is limited knowledge about most species and their venoms, in part due to the technical and welfare challenges associated with venom extraction, low venom yields, and the lack of cellular models available. Organoids represent an excellent opportunity to overcome these challenges. Here, we establish, for the first time, venom gland organoids from snakes of the Colubridae family and demonstrate the in vitro production of toxins.

Antivenom administration is currently the only therapy for snakebite envenoming. However, in sub-Saharan Africa, inadequate quality control systems have led to deficits in the availability, accessibility, efficacy and safety of regionally available antivenoms, which, in turn, hinder snakebite treatment and management in the region. To address this impediment to snakebite treatment in Kenya, this study aimed to assess the preclinical neutralising potencies of four different antivenoms previously or currently available in Kenya (SAIMR polyvalent, AFRIVEN, PANAF-Premium^TM and Inoserp^TM) against key snakes of medical importance in the region, towards establishing a national antivenom quality control laboratory. Venoms were extracted from the Kenyan “big five” medically important snake species: Naja ashei, Naja pallida, Naja nigricollis, Dendroaspis polylepis and Bitis arietans, and their lethal potencies were determined using a murine median lethal dose (LD₅₀) assay. In vitro immunological assays (ELISAs and immunoblotting) and an established preclinical murine in vivo neutralisation assay (median effective dose [ED₅₀]) were used to assess the immunoglobulin-binding and venom-neutralising efficacies of the test antivenoms. In vitro assays revealed high venom-binding titres of SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM, and reactivity to a wide range of venom proteins across the different snake venoms. Contrastingly, Inoserp^TM antivenom had low binding titres and poor reactivity to the snake venom proteins. These findings were aligned with the in vivo results, where SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM showed potent venom-neutralising efficacies against all the tested snake venoms, while Inoserp^TM had low potency and failed to neutralise the lethal effects of N. ashei, N. pallida and D. polylepis venoms at the manufacture-claimed doses. Based on these robust preclinical results, we conclude that SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM antivenoms offer considerable potential for the treatment of envenoming by diverse medically important snakes in Kenya. The observed deficiencies with the Inoserp^TM product highlight the importance of (i) robust, independent preclinical antivenom efficacy testing and (ii) the value of establishing a quality control laboratory to inform local regulatory and procurement decision making.

Snakes are a valuable yet understudied taxon for investigating evolutionary adaptations in the vertebrate retina. They possess up to three visual pigments: a short‐wavelength‐sensitive opsin (SWS1), a medium/long‐wavelength‐sensitive opsin (LWS), and rhodopsin (RH1). Nocturnal snakes have duplex retinas containing both rod and cone photoreceptors, whereas diurnal caenophidian (“advanced”) snakes exhibit simplex “all‐cone” retinas, lacking morphologically typical rods. In this study, we analyzed photoreceptor morphology in the retinas of caenophidian snakes using high‐resolution scanning electron microscopy (SEM) and examined visual‐opsin expression patterns with immunohistochemistry (IHC). Our analyses revealed remarkable interspecific variability in visual‐cell morphology. Light microscopy showed that in all sampled diurnal caenophidians, photoreceptors expressing RH1 exhibit a gross cone‐like morphology. However, SEM analysis revealed a subset of photoreceptors with distinct features—thinner inner segments and rod‐like synaptic terminals—suggesting they are transmuted, cone‐like rods. In retinal sections from nocturnal caenophidian snakes, coexpression of the cone opsins SWS1 and LWS in individual cones was observed, whereas rhodopsin expression remained restricted to morphologically typical rods and showed no coexpression. In contrast, diurnal caenophidians commonly coexpress rhodopsin and SWS1 in single cones, with some instances of triple coexpression (SWS1, RH1, and LWS) in single cones. We evaluated the patterns of spatial distribution of RH1‐ and SWS1‐expressing photoreceptors, as well as SWS1 + RH1 multiopsin cones, in wholemounted retinas of ten species. Our findings revealed considerable species‐specific variation in photoreceptor density, topography, and opsin coexpression patterns. IHC results suggest that in some species, rhodopsin is not only expressed in transmuted, cone‐like rods but may also be co‐opted by UV/violet‐sensitive (SWS1‐expressing) cones. These findings underscore the exceptional diversity and adaptive innovation in snake visual systems. The unique features and striking interspecific differences in their photoreceptors highlight snakes as an outstanding taxon for studying vertebrate visual‐system function and evolution.

Identification and characterization of snake venom toxins that interfere with hemostasis have important implications for the treatment of snake envenomation, the bioprospecting of therapeutically useful molecules, and the development of research tools for investigating hematologic disorders. Many venoms have been shown to possess thrombolytic activity. However, it remains unclear if actions on other clot-stabilizing proteins beyond fibrin chains contribute significantly to venom-induced thrombolysis because the clot-wide targets of venom proteases and the mechanisms responsible for thrombolysis are not well understood. Here, we utilize a high-throughput time-based thrombolysis assay in combination with untargeted peptidomics to provide comprehensive insight into the effects of venom from six snake species on blood clot degradation. We compare thrombolytic profiles across venoms with variable levels of proteases and generate venom-specific fingerprints of cleavage specificity. We also compare the specific effects of venoms that possess a range of thrombolytic activity on fibrin subunits and other clot-bound proteins involved in clot structure. Venoms with higher thrombolytic activity demonstrated an enhanced ability to target multiple sites across fibrin chains critical to clot stability and structure, as well as clot-stabilizing proteins including fibronectin and vitronectin. Collectively, this study significantly expands our understanding of the thrombolytic and fibrinolytic effects of snake venom by determining the full suite of clot-specific venom targets that are involved in clot formation and stability.

Mosquitoes are responsible for the transmission of numerous pathogens, including Plasmodium parasites, arboviruses and filarial worms. They pose a significant risk to public health with over 200 million cases of malaria per annum and approximately four billion people at risk of arthropod-borne viruses (arboviruses). Mosquito populations are geographically expanding into temperate regions and their distribution is predicted to continue increasing. Mosquito symbionts, including fungi, bacteria and viruses, have desirable traits for mosquito disease control including spreading horizontally and vertically through mosquito populations and potentially colonising multiple important vector species. Paratransgenesis, genetic modification of mosquito symbionts with effectors to target the pathogen rather than the vector, is a promising strategy to prevent the spread of mosquito-borne diseases. A variety of effectors can be expressed but venom toxins are excellent effector candidates because they are target specific, potent, and, stable. However, the only toxins to be explored in mosquito paratransgenesis to date are Scorpine and mutated phospholipase A2. To enhance the scope, effectiveness, and durability of paratransgenesis, an expanded arsenal of effectors is required. This review discusses other potential toxins effectors for future paratransgenesis studies based on prior in vitro and in vivo antiparasitic and antiviral studies and highlights the need for further research and investment in this area. In terms of mosquito-borne diseases, paratransgenesis strategies have been developed to target Plasmodium. We postulate the potential to apply this principle to target arboviruses using antiviral toxin effectors.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.