Arthropod-borne viruses (arboviruses) transmitted by mosquitoes are an ever-present burden to human health, with flaviviruses alone responsible for ~400 million global infections annually. The expansion of vector distributions, emergence of epizootic arboviruses, growing incidence of insecticide resistance and increased international travel, all threaten to outstrip current vector control strategies. The release of benign, commensal, insect-specific viruses (ISVs) that can induce superinfection exclusion of arboviruses in mosquitoes may be a novel means of arboviral control. Despite promising data in vitro, the in vivo demonstration of superinfection exclusion and the understanding of the molecular mechanisms behind this phenomenon remains limited. This project aims to further the understanding of superinfection exclusion by; (1) Establishing lines of mosquitoes persistently infected with select Australian ISVs; (2) Testing the capacity of these ISVs to exert superinfection exclusion against key arboviruses in vivo; and (3) Utilising RNAseq to identify putative genes and RNA interference pathways involved in superinfection exclusion. We present quantitative host range and tissue tropism data on the ISVs: Binjari virus, Castlerea virus, Palm Creek virus and Parramatta River virus; and how these relate to the potential for superinfection exclusion. We also present the discovery of a novel mosquito ISV infection route and the implications that this has for our understanding of ISV transmission cycles and the inoculation of mosquitoes with beneficial ISVs en masse.