Emerging infectious diseases (EID) significantly impact public health and have the potential to cause pandemics. Outbreaks of EID have increased in recent decades, with RNA viruses responsible for a substantial proportion of them. Outbreaks are more likely to occur in areas with high biodiversity, poor sanitation and public health infrastructure, and limited resources for EID control. Furthermore, clinical symptoms of many viral infections overlap, making them challenging to diagnose correctly. Metagenomic sequencing (metagenomics) negates the need for targeted detection assays, generates virus genome information and can detect novel or genetically divergent viruses. Through combining targeted PCR-based assays with untargeted metagenomics, this project aimed to detect infections in two cohorts of patients with fever from low-and-middle income countries (Sierra Leone and Ecuador), characterise any viruses identified and pursue further knowledge relevant to EID. Plasmodium, Leptospira and Ebola virus (EBOV) infections were detected in Sierra Leonean patient samples using PCR-based assays. Human immunodeficiency virus, GB virus C (human pegivirus) and hepatitis B virus were identified in 36 PCR-negative Sierra Leonean patient samples using metagenomics. The detection of EBOV was surprising because the patients tested negative for EBOV RNA using a qRT-PCR assay at the time of sampling; further investigation suggested the discrepancies were related to assay sensitivity. This project detected and isolated Oropouche virus (OROV), an emerging arbovirus, from a patient from Ecuador for the first time. Metagenomics revealed that the Ecuadorian strain was divergent from other strains at an established diagnostic qRT-PCR binding site. This information allowed the optimisation of a qRT-PCR assay, which subsequently identified further OROV infections within the patient cohort. Adoption of this assay in relevant countries could enhance OROV surveillance and diagnosis. This demonstrates the value of using metagenomics alongside PCR-based assays in screening studies to ensure diagnostic assays can detect current strains. In addition to OROV, Dengue virus, Hepatitis A virus, Zika virus, and Leptospira were identified in Ecuadorian patient samples. Phylogenetic analyses of OROV sequences suggested that an OROV outbreak occurred in Esmeraldas, Ecuador in 2016. OROV vector Culicoides paraensis is not present in this area, raising the question of alternative insect vectors. Experiments demonstrated that OROV replicates in Aedes spp. cell lines. These species are important mosquito vectors of other arboviral diseases and this finding warrants further investigation. Further experimental work identified human fibroblasts and hepatocytes as potentially relevant to OROV pathogenesis in humans.

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