Flaviviruses usually cycle between arthropod vectors and vertebrate hosts, however a recently identified group of insect-specific flaviviruses (ISFs) exhibit transmission only between mosquitoes. To investigate the diversity and prevalence of ISFs in Australia, mosquito samples were screened by an ELISA-based virus detection system targeting viral dsRNA intermediates produced in inoculated mosquito cell cultures. ELISA-positive samples were then identified using a generic flavivirus RT-PCR. A high proportion of Aedes vigilax mosquitoes collected from Sydney in 2007 yielded isolates of a novel species of ISF most closely related to an ISF from Finland. PaRV has subsequently been isolated from mosquitoes collected from Sydney, Newcastle and Brisbane, indicating it has persisted in Ae. vigilax populations and exhibits a wide geographic distribution in Australia.
PaRV failed to replicate in a range of vertebrate cell lines and was restricted to growth in mosquito cells derived from Aedes species. When replication kinetics were examined in Ae albopictus cells PaRV replicated to 50-fold higher titres than West Nile virus(WNV) within the first 24h but by day 5 WNV produced 500-fold more virus than PaRV. This trend was consistent in both RNAi-deficient and RNA-competent cells suggesting that PaRV has rapid initial replication in mosquito cells but is then regulated by an RNAi-independent mechanism that may be important for maintaining persistent infection. The mode of PaRV transmission between mosquitoes was assessed using progeny of naturally-infected wild female mosquitoes collected from Sydney. PaRV was isolated from a high proportion of both male and female progeny suggesting that this virus is efficiently maintained by vertical transmission. Preliminary in vitro studies have shown that PaRV can suppress the early stage replication of WNV in co-infected mosquito cell culture. Further studies to determine if PaRV can influence vector competence of Ae. vigilax for pathogenic viruses are currently being undertaken.