Fig 2. Generation and characterization of 75A.stop and ORF75B.stop viruses.
(A) Schematic of the 75A.stop mutations in MHV68. Restriction sites used to verify stop codon insertions are underlined. (B) Restriction fragment length polymorphism (RFLP) analysis of 75A.stop mutants after BamHI digestion. (C) Single-step growth curve of 75A.stop mutants and parental H2B-YFP viruses in NIH 3T3 fibroblasts (MOI 5). (D) Schematic of the ORF75B mutations in MHV68. (E) RFLP analysis of ORF75B.stop mutants after EcoRI digestion. (F) Single-step growth of ORF75B.stop mutants and marker rescue virus in NIH 3T3 fibroblasts (MOI 5). Error bars represent +/- SD of triplicate infections. C57B/6 mice were inoculated with 1000 PFU via the intranasal route with the indicated viruses. Lung homogenates from mice were titered by plaque assay. (G) Timecourse analysis of acute replication on the lungs. (H) Acute replication in the lungs 7 dpi. Each symbol represents an individual mouse. Line indicates geometric mean titer. The dashed line depicts the limit of detection at 50 PFU/ml of lung homogenate (log10 of 1.7). * p ≤ 0.05; ** p ≤ 0.005; **** p ≤ 0.0005; for G-H, significance determined by one-way ANOVA analysis.