FIG 1.

Recovery of UUKV from Pol I-driven plasmid DNAs. (A) The trisegmented, negative-sense RNA genome of UUKV. The black arrowhead shows the cleavage site in the polyprotein precursor of the glycoproteins G_N and G_C. The black bars indicate the nucleotides found to be mutated in the UUKV laboratory strain relative to the sequence of UUKV strain 23. The penetrance of each mutation is indicated underneath. Four to five clones were analyzed for each viral genome segment. (B) Schematic representation of the Pol I-driven UUKV rescue system. (C) Focus-forming assay used for the titration of UUKV strains. Examples are shown for the UUKV lab strain (UUKV) and the viruses rescued from plasmid DNAs after five passages in BHK-21 cells (rUUKV and rUUKV S23). After 3 days of incubation at 37°C, foci were immunostained with the rabbit polyclonal antibody U2 against the viral proteins N, G_N, and G_C. (D) rUUKV and rUUKV S23 production 5 days after transfection of plasmids expressing L, M, and S segments under the control of the Pol I promoter together with the UUKV L and N expression plasmids pUUK-L and pUUK-N in BHK-21 cells. (E) Titer of rUUKV and rUUKV S23 after rescue (passage 0, P0) and up to five passages (P1 to P5) in BHK-21 cells. FFU, focus-forming units. (F) Sequence analysis of the rUUKV S23 M segment compared to that of rUUKV carried out from vRNA purified extracts after five passages in BHK-21 cells.