Arboviruses, including medically-relevant flavivirus pathogens such as dengue, Zika, and West Nile viruses, are transmitted to humans and other vertebrate species primarily by mosquitoes. There is a subset of flaviviruses known as Insect-specific flaviviruses (ISFs), that replicate only in mosquito cells and can be maintained in some mosquito populations at high prevalence by efficient vertical transmission (VT) from female mosquito to their progeny. There is increasing evidence that mosquitoes that carry ISFs have a reduced capacity to transmit pathogenic flaviviruses. This phenomenon, known as superinfection exclusion, could offer a novel, natural, and environmentally friendly method for arbovirus biocontrol.
To fully exploit ISFs as a biocontrol strategy, further understanding of the viral factors that contribute to the efficiency of their vertical transmission and high prevalence in some mosquito populations, and the mechanisms of superinfection exclusion is required. However, there is currently a lack of research tools, such as infectious virus clones and virus specific antibodies, to undertake this research. In this project we constructed cDNA clones of novel Australian isolates of two ISFs, Culex Flavivirus (CxFV) and Cell fusing agent virus (CFAV), found in Culex and Aedes species respectively by elucidating their complete genome sequences and using Circular Polymerase Extension Reaction (CPER) to produce wild type and genetically modified forms of these virus. This reverse genetics system has also enabled the construction of viable chimeric viruses with genetic components of both the CxFV and WNV, to study the contribution of individual viral factors to VT. We also used purified preparations of CxFV and CFAV to immunise mice to produce virus-specific polyclonal and monoclonal antibodies to identify the tissues/organs infected by ISF during vertical transmission. The research tools generated in this project will be essential to characterize the biology of ISFs and their potential as novel arbovirus control agents.