FIG 2.

Creation of OROV mutant viruses. (A) Schematic of the M segment showing Gn, NSm, and Gc regions. The arrows depict where cleavage occurs. The patterned box indicates the signal peptide, and the black boxes represent transmembrane domains. Nucleotides 1036 to 1475 were deleted in order to generate delNSm M segment. (B) S segment products N and NSs. NSs is coded from an overlapping reading frame with N. Schematic shows how NSs mutants differ from the wt. rOROV246NSs has a stop codon (asterisk) placed at nucleotide (nt) position 314 of S segment cDNA, changing TTA to TAA and thereby deleting the last 8 aa. rOROVdelNSs has a stop codon at cDNA nt position 116, changing TGG to TAG so that a stop codon is generated immediately after the second start codon (methionine [M]). Numbers are amino acid lengths. (C) rOROV2080S S segment in comparison to wtOROV and rOROV S segment. Numbers are nucleotide positions. Arrows show where changes occur. First two positions generate a variation in the NSs OROF. Black highlights the UTRs. (D) Growth properties of recombinant viruses in Vero E6 cells. Cells were infected at an MOI of 0.1. Samples were harvested at the indicated time points and titrated on BHK-21 cells. The graph shows results of a representative experiment. (E) Plaque phenotype of recombinant viruses in BHK-21 cells. A plaque assay was carried out, and at 3 days p.i., cells were fixed and stained with crystal violet. (F) N production in recombinant viruses. Cell lysates from the growth curve (D) were probed for OROV-N and tubulin.