Novel replication-competent reporter-expressing Rift Valley Fever Viruses for molecular studies

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic disease that causes severe disease in both domestic and wild ungulates and humans, making it a significant threat to livestock and public health. The RVFV genome consists of three single-stranded, negative-sense RNA segments differing in size: Small (S), Medium (M) and Large (L). Segment S encodes the virus nucleoprotein N and the virulence-associated factor non-structural (NSs) protein in opposite orientations, separated by an intergenic region (IGR). To overcome the current need of using secondary techniques to detect the presence of RVFV in infected cells, we used T7-driven polymerase plasmid-based reverse genetics to generate replication-competent recombinant (r)RVFV expressing Nanoluciferase (Nluc) or Venus fluorescent proteins. These reporter genes were used as valid surrogates to track the presence of RVFV in mammalian and insect cells. Notably, we explored the genome plasticity of RVFV and compared four different strategies by modifying the viral segment S in order to introduce the reporter gene foreign sequences. The reporter-expressing rRVFV were stable and able to replicate in cultured mammalian and insect cells, although to a lesser extent than the recombinant wild-type (WT) counterpart. Moreover, rRVFV expressing reporter genes were validated to identify neutralizing antibodies or compounds with antiviral activity. In vivo , all mice infected with the reporter-expressing rRVFV displayed an attenuated phenotype, although at different levels. These rRVFV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of RVFV, and represent an excellent biotechnological tool for developing new therapeutics against RVFV infections.