Creation of Rift Valley Fever Viruses with four-segmented Genomes reveals flexibility in Bunyavirus Genome Packaging.

Bunyavirus genomes comprise a small (S), medium (M) and a large (L) RNA segment of negative polarity. Although the untranslated regions (UTRs) have been shown to comprise signals required for transcription, replication and encapsidation, the mechanisms that drive the packaging of at least one S, M and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). Here we studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse-genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising 2 S-type segments in the absence of an M-type segment to a virus consisting of 4 segments (RVFV-4s) of which 3 are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines