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The leishmaniases are a complex group of vector-borne diseases caused by protozoan parasites of the genus Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatidae) and are transmitted between vertebrate hosts by phlebotomine sandflies. These diseases contribute to a significant One Health burden globally. In Australia, the finding of a novel Leishmania (Mundinia) macropodum Barratt, Kaufer & Ellis, 2017 with evidence of a distinct transmission dynamic, has raised interesting questions. In contrast to other Leishmania species, day-biting midges Forcipomyia (Lasiohelea) Kieffer, 1921 have been suspected as the vector responsible for transmitting cutaneous leishmaniasis (CL; a disease affecting the skin) to marsupial species in the Northern Territory (NT). In addition to an unidentified vector, the life cycle of L. macropodum has so far been based on assumptions with no knowledge of other possible hosts (in addition to macropods) and geographical range. The findings described in this thesis contribute new information on the transmission dynamics of L. macropodum, with a particular focus on the interplay between vector biology, parasite characteristics, and host immune factors. Specifically, this thesis i) examined and characterised the natural exposure of L. macropodum in humans and non-human vertebrate hosts in an Australian region of Leishmania endemicity within marsupial populations, and ii) assessed the competence of the incriminated vector of L. macropodum transmission., Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Full Text

Arboviruses circulating in Australia are of clinical importance as they cause painful, often chronic musculoskeletal arthritic or fatal dengue disease. They include Ross River virus (RRV), Barmah Forest virus (BFV), chikungunya virus (CHIKV) and dengue virus (DENV). This dissertation assessed the seroprevalence, diversity, evolution, spread and strategies for controlling infection caused by these arboviruses. The overall aim of research presented in this dissertation was to improve public health responses such as nationwide surveillance, diagnosis, and infection control. Research undertaken in chapter 2 achieved this through systematic review and statistical synthesis of human seroprevalence data for RRV, BFV and DENV. I have shown that many factors influence the reporting of human seroprevalences for these three arboviruses. Among these factors is the choice of assay method, timeframe, and sampling strategy. I have also presented findings showing that the spatiotemporal range and median seroprevalences reported for DENV and RRV are wider and higher than those for BFV. This indicates that the risk of exposure to DENV and RRV is significantly higher and extends overseas, however this risk relates to the distribution of the transmitting vectors. My analysis highlighted that DENV presents the highest risk of exposure but its incidence in Australia is declining due to successful implementation of Wolbachia-DENV control programs which have restricted the distribution of Aedes aegypti. Most serosurveys reviewed reported a positive association between age and seroprevalence, and increasing seroprevalence with gender. However, studies reporting gender-seroprevalence data did not do statistical analysis to show the significance of outcomes. This dissertation therefore recommends streamlining, standardization and statistical synthesis of serosurveys data to better quantify risk of exposure, identify risk factors, at-risk groups/populations, associations between seroprevalence, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Griffith Health, Full Text

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Griffith Health, Arthritogenic alphavirus infection causes debilitating pain in the joints and muscles with many patients experiencing such symptoms chronically. However, there is insufficient evidence to explain the underlying causes behind symptoms of persistent arthralgia and myalgia. Joint-associated tissues are the main site of inflammation during alphavirus infection, and it has been shown that alphaviruses induce damage to the cartilage and synovium. Therefore, the cell types present in these tissues play critical roles in disease pathogenesis. The findings described in this thesis contribute to the general understanding of host-pathogen interactions during alphavirus infection of joint-associated cell types. Here, the analysis of murine joints revealed chondrocytes as a target of RRV infection (Chapter 1). Further evaluation of human primary chondrocytes and skeletal muscle cells through short-term in vitro cell culture showed that these cell types could support productive RRV infection. Our study presents the first evidence of the role of chondrocytes in alphavirus disease pathogenesis. Currently, there are gaps in our understanding of chronic alphavirus disease, especially in the absence of detectable viraemia after recovery from infection. Here, we have investigated the r sponses of several cell types in joint-associated tissues during chronic infection. Human primary cells and their corresponding cell line counterparts for chondrocytes, muscle cells and fibroblast-like synoviocytes (FLS) were infected with four alphaviruses of clinical importance, namely Ross River virus (RRV), Barmah Forest virus (BFV), chikungunya virus (CHIKV) and o’nyong’nyong virus (ONNV). We found that all cell types studied were able to retain residual alphaviral nucleic acids after recovery from infection despite several passages in culture (up to 10 weeks), indicating the potential of these cell types as reservoirs for the virus and/or viral RNA (Chapter 2). Regretfully, we were unable to determ

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Griffith Health, Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Current vector control strategies have been impeded by mosquitoes acquiring resistance to insecticide. Therefore, development of new vector control strategies is urgently needed to complement current strategies. In this thesis, efforts have focused on characterizing the glycan-lectin interactions of Ross River virus (RRV; Togaviridae: Alphavirus) with their mosquito vectors. RRV is the most common arbovirus in Australia that causes clinical manifestations including arthralgia and myalgia. Many studies have shown the importance of viral surface glycans in mediating viral entry into host cells. Moreover, the viral surface glycans varies depending on the cells that they replicate in and this variation can affect the infectivity of virus. However, gaps remain in the role of viral glycans in virus host cell recognition. In Chapter 2, the surface glycans of RRV derived from two different cell lines, C6/36 (Ae. albopictus) and Vero (African green monkey kidney) were characterized using lectin array. Lectin array data revealed that RRV derived from two different cell lines exhibited similar glycan profiles. The glycan structures present on the surface of RRV are mannose, galactose, N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). To investigate the importance of these viral surface glycans in mediating viral entry into host cells, six lectins targeting these glycan structures were assessed for their ability to bind and block RRV entry into host cells. Of these lectins, two mannose-binding lectins, GNA and ConA, showed inhibition of RRV entry into C6/36 and Vero cells. These results suggest the potential use of mannose-binding lectins to block RRV transmission by mosquitoes. It is well known that cell surface glycans or lectins play important role in viral entry. Therefore, comprehensive surface glycan profiles and carbohydrate-binding characteristics o

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Environment and Sc, Science, Environment, Engineering and Technology, Arboviruses contribute a significant burden to human and animal health. Circulation of arboviruses comprises three components; blood-sucking arthropods, vertebrate hosts, and viruses that can infect vertebrates and invertebrates. Interaction of these components is dependent on ecological factors (such as species distributions and climate), epidemiological factors (including vector and host immunity) and behavioural determinants (such as vector feeding host preference or host defensive behaviours). Identifying these drivers of disease emergence can be complicated but informs efforts to mitigate on-going circulation. This dissertation generates new perspectives on the transmission dynamics of Australia’s most common arbovirus, Ross River virus (RRV), with a particular focus on non-human reservoirs. Specifically, I (a) critically analysed current and historic knowledge for non-human reservoirs and vector feeding patterns, (b) examined the natural exposure of RRV in human, free-living and domestic vertebrate populations, and (c) assessed the vertebrate and vector community ecology across areas of varying human notification rates. I began by undertaking a systematic literature review (Chapter 2) assessing evidence for non-human reservoirs of RRV. This chapter synthesised published serological, virus isolation and experimental infection studies in light of the long-held dogma that marsupials are the primary reservoir of RRV. A key finding of this chapter was emerging evidence that placental mammals and birds were also capable of transmitting RRV to mosquito vectors, suggesting a broader reservoir potential than marsupials alone. To further assess the current and historic knowledge, a meta-analysis was performed on mosquito blood meal analysis studies (Chapter 3). It was evident from this chapter that Australian mosquitoes have highly varied feeding patterns which did not reflect their taxonomic classification or larval ecology. To understand the natural exposure of RRV in

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Institute for Glycomics, Griffith Health, Arthritogenic alphaviruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) are transmitted by mosquito vectors, and can cause musculoskeletal disease manifestations such as, excruciating pain and inflammation in joints and the surrounding muscle tissues, in infected individuals. Despite the recent findings of cartilage damage, erosion and focal necrosis in murine models of both chikungunya viral disease (CHIKVD) and Ross River viral disease (RRVD), the role of human chondrocyte/cartilage in alphaviral infection has never been previously explored. In this thesis, it was shown that, both the lower and the higher density cultures (high-density cell cultures and micromass pellet cultures) of primary chondrocytes, supported the RRV-T48 infection and produced the upregulation of soluble inflammatory host factors during the infection. Interestingly, the heterogeneity of chondrocyte infectibility was also noted between two human chondrocyte donors. Chondrocytes that were derived from one patient could not sustain an efficient RRV-T48 replication when compared to chondrocytes that were isolated from another patient. Although this infection profile was surprising, the sample pool for the donor chondrocytes was extremely small thus, other confounding factors may have also contributed to the difference in infection. Additionally, the presence and co-localisation of RRV antigens within the chondrocytes of murine joints, proved that RRV could get to the cartilage and either cause damage directly or trigger the body systems to potentiate the damage. The second half of the thesis focused on exploring a new class of heparan sulfate (HS)-modulating compounds, as targeted treatments for arthritogenic alphaviral infection/diseases. Current drugs for alphaviral diseases are non-specific and include the use of non-steroidal anti-inflammatories which only provide temporary or partial relief in patients. Recently pentosan polysulfate (PPS), a sulfated polysaccharide and a HSmi

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.