The genus Flavivirus encompasses a diverse array of viruses including West Nile virus (WNV), dengue viruses and Japanese encephalitis virus that are responsible for significant mosquito-borne disease around the world. These arboviruses are transmitted between mosquitoes and vertebrates, relying on replication in both hosts for maintaining their natural transmission cycle. However over the last decade a new subgroup of insect-specific flaviviruses (ISF) have been identified which are restricted to replicating only in mosquito hosts. Of particular interest is the observation that transmission of pathogenic viruses, such as WNV, is reduced in mosquitoes persistently infected with ISFs. Recently our lab discovered a novel ISF from Aedes vigilax mosquitoes in Sydney named Parramatta River virus (PaRV).
This study aimed to use a novel technique known as circular polymerase extension cloning (CPEC) to generate infectious clones of PaRV, the Kunjin strain of West Nile virus (WNVKUN) and chimeras derived from both viruses to identify the genes responsible for host restriction in ISFs. To date we have successfully generated a WNVKUN infectious cDNA clone, using a modified insect promoter (OpIE2) to drive transcription of infectious viral RNA in transfected mosquito cells. This is the first report of using the CPEC system to generate an infectious cloned flavivirus in mosquito cells. A similar approach is now being used to generate infectious clones of PaRV and PaRV/WNVKUN chimeras. These viral constructs will offer a unique opportunity to elucidate the molecular basis of the restriction of ISF to mosquito hosts and provide novel insights into the evolution of flaviviruses in general. Manipulation of infectious ISF clones may also provide a novel strategy to develop biological agents to suppress the transmission of vertebrate-infecting flaviviruses (VIF) by mosquitoes.