Generation of an infectious cDNA clone of Okushiri virus and its derivative capable of expressing an exogenous gene

The Okushiri virus (OKV), isolated from Aedes larvae collected on Okushiri Island, Hokkaido, Japan, is a member of the Negevirus family, a newly proposed insect-specific virus group (genus). To enable genetic manipulation of the genome, we constructed an infectious cDNA clone of OKV. RNA synthesized in vitro from pFBOKV, a full-length OKV cDNA, in the presence or absence of cap analogue produced infectious progeny viruses effectively in mosquito C6/36 cells. Subsequently, ORF3 in pFBOKV was replaced with a GFP coding sequence to generate a construct designated as pO2GFP. C6/36 cells transfected with pO2GFP-derived RNA successfully expressed GFP, but failed to produce progeny viruses. Co-transfection of C6/36 cells with pO2GFP- and pFBOKV-derived RNA revealed that it is possible to produce infectious pO2GFP-derived progeny viruses by supplying OKV genetic elements and/or gene product deleted in pO2GFP even though the infectivity appeared to be low. Although improvements are required to construct viral vectors that propagate more efficiently, this is, to our knowledge, the first report of construction of a negevirus-based foreign gene expression system. The cDNA clones constructed and biological insights obtained in this study will be powerful tools allowing for reverse genetics of OKV and providing a basis to develop efficient negevirus-based expression vector systems.