FIG 2.

Generation of VP4 and NSP3 mutant viruses using plasmid-only based reverse genetics system. (A) Schematic representation of a 13-plasmid system for generation of mutant viruses expressing spike epitopes (adapted from Kanai et al. [42] and Komoto et al. [43]; created with BioRender.com). Full-length cDNAs representing each of the 11 gene segments were transfected into BSR-T7 cells with increasing amounts of two plasmids carrying NSP2 and NSP5 genes, along with two plasmids expressing vaccinia virus capping enzyme genes (D1R and D12L). Mutant viruses were rescued following amplification in MA104 cells. (B) Multistep growth curves comparing the bovine rotovirus (RV) RF isolate with reverse genetics (RG) virus. *, P ≤ 0.05 (24 hours postinfection [hpi], P = 0.0473; 48 hpi, P = 0.0407). ***, P ≤ 0.001 (32 hpi, P = 0.0005). Data are from three independent experiments. Dashed line represents detection threshold. Viral titers and plaque morphology versus WT for VP4 (C and D) and NSP3 (E and F) mutants. Dashed line represents detection threshold. (C) *, P ≤ 0.05 (RBM.1, P = 0.0205; RBM.2, P = 0.0217). **, P ≤ 0.01 (HR2, P = 0.0018). Representative results from three independent rescues are shown (except for RBD, which only rescued once). In panel E, open circles show failed rescues plotted at the limit of detection.