Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are responsible for significant mosquito-borne disease. However recently, insect-specific flaviviruses (ISF) which only replicate in mosquito hosts have been identified. Of particular interest, mosquitoes persistently infected with some of these ISFs are less capable of transmitting pathogenic flaviviruses . This study aims to generate infectious clones and chimeras of the Australian ISF, Parramatta river virus (PaRV) and WNV to identify the genes responsible for host restriction in ISFs.
A novel technique known as circular polymerase extension cloning (CPEC) was used to generate an infectious clone from fragments of viral cDNA. A modified CMV or OpIE2 promoter was used to drive transcription in either HEK293 mammalian or C6/36 insect cells, respectively. Viral constructs were transfected into their respective cell line and characterised using a combination of IFA, rt-PCR and ELISA.
This study successfully generated infectious clones of WNV and PaRV, using a modified OpIE2 insect promoter in transfected C6/36 mosquito cells. Viral authenticity was confirmed using sequencing and antibodies specific to each of the viruses. Similar approaches are currently being utilised to generate chimeric constructs of PaRV and WNV.
These constructs offer a unique opportunity to elucidate the mechanisms restricting ISFs to mosquito hosts and provide novel insights into flaviviral evolution. Manipulation of infectious ISF chimeras may also provide novel strategies to suppress the transmission of vertebrate-infecting flaviviruses by mosquitoes and provide new generation platforms for the safe production of diagnostic antigens and vaccines against pathogenic flaviviruses.