We recently discovered several insect-specific flaviviruses (ISFs) in mosquitoes from different regions of Australia. Based on our previous observations that ISFs fail to replicate in vertebrate cells, we undertook a thorough investigation of these viruses to further define their host range, the molecular mechanisms of their host restriction and their potential as tools for biotechnology.
Determination of the complete genome sequences of these viruses and phylogenetic analysis revealed we had discovered 7 new ISF species representing two distinct genetic lineages. As well as confirming their inability to infect a range of vertebrate cell lines, we also observed variation in their mosquito host range by inoculating cells derived from different mosquito genera and detecting replication by the appearance of viral proteins, viral replicative RNA and siRNA responses by the host cell. Surprisingly, some viruses were restricted to a single mosquito species. Construction of infectious DNAs and chimeric viruses also allowed us to identify stages at pre- and post-cell entry where virus infection and replication is blocked in the infection cycle of vertebrate cells.
Using this approach we also generated a series of chimeric viruses expressing the structural genes (prM-E) from several pathogenic flaviviruses spliced into the genetic backbone of two different ISF species. Along with a new panel of ISF-specific monoclonal antibodies recently prepared in our lab, these chimeric viruses provide a unique set of tools to further define the mechanisms of ISF host restriction and for assessment as novel reagents in biotechnology.