FIG 1.

Characterization of recombinant OROV. (A) RT-PCR products derived from the L, M, and S segments of OROV strain BeAN19991. Amplified products were separated on a 1% agarose gel along with a 1-kb marker (Promega). Products were cloned into plasmids containing a T7-RNA polymerase promoter and a hepatitis delta ribozyme as shown in the schematic at the bottom. (B) Growth properties of wild-type (wt) and recombinant (r) OROV in Vero E6 cells. Cells were infected at an MOI of 0.1. At indicated time points samples were harvested and titers determined by plaque assay on BHK-21 cells. The graph shows results of a representative experiment. (C) Western blot showing N protein synthesis from the growth curve (A). Tubulin was probed as a loading control. (D) Comparison of plaque phenotypes of wtOROV and rOROV. A plaque assay was carried out on BHK-21 cells, and at 3 days p.i., cells were fixed and stained with crystal violet. (E) Effects of different MOIs on rOROV yields in Vero E6 cells. Infected cells were harvested 48 h p.i. and titrated on BHK-21 cells. A graph is presented for a representative experiment. (F) Comparison of rOROV growth in various cell lines. Indicated cells were infected at an MOI of 0.001 and at 48 h p.i. harvested and titrated on BHK-21 cells. Bars represent ranges from two experiments. (G) Comparison of rOROV plaque phenotypes on BHK-21, Vero E6, A549, and CPT-Tert cells. Infected cells were fixed and stained with crystal violet at 3 days p.i.