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. Author manuscript; available in PMC: 2016 Jan 15.
Published in final edited form as: J Immunol. 2014 Dec 8;194(2):678–689. doi: 10.4049/jimmunol.1401833

Figure 2. Recombinant RRV-LCMV is similar to WT RRV in vitro and in vivo.

Figure 2

(A) Schematic of recombinant RRV-LCMV virus encoding the CD8 (gp33) and CD4 (gp61) immunodominant epitopes of the LCMV glycoprotein. (B) BHK-21 cells were inoculated with a MOI of 0.01 PFU/cell of WT RRV or RRV-LCMV, and infectious virus in the supernatant was titered at 0 (inoculum), 1, 3, 6, 12, and 24 hpi by direct plaque assay on BHK-21 cells. Three-to-four week-old C57BL/6J WT mice were mock-inoculated (PBS) (n = 3) or inoculated with 103 PFU of WT RRV (n = 7) or RRV-LCMV (n = 15) and assessed for (C) weight gain and (D) musculoskeletal disease signs including loss of gripping ability and altered gait at 24-hour intervals. Each data point represents the arithmetic mean ± SEM. * P < 0.05 for days 7−10 pi between WT RRV and RRV-LCMV groups as determined by two-way ANOVA followed by a Bonferroni multiple comparison test. (E, F) Three-to-four week-old C57BL/6J WT mice were or inoculated with 103 PFU of WT RRV or RRV-LCMV, and RRV genome levels in the right quadriceps muscle (E) and right ankle/foot (F) were analyzed by absolute RT-qPCR at 5 dpi (n = 4) and 10 dpi (n = 5). Horizontal bar represents the mean. * P = 0.02, ** P = 0.009, as determined by two-way, unpaired t-tests. (G) Five-μm paraffin-embedded sections were generated from the gastrocnemius muscles of mock (n = 2), WT RRV (n = 4), and RRV-LCMV (n = 5) infected mice at 10 dpi and H&E stained. (H) H&E-stained sections from 10 dpi were scored in a blinded manner for the degree of inflammation based on the following scale for the percentage of tissue affected: 0, absent (0%); 1, minimal (<10%); 2, mild (11–25%); 3, moderate (26–40%); 4, marked (41–60%); and 5, severe (>60%). Graphs represent the arithmetic mean ± SEM. (I) Three-to-four week-old C57BL/6J WT mice were mock-inoculated (n = 6) or inoculated with 103 PFU of WT RRV (7 dpi, n =7; 10 dpi, n = 11) or RRV-LCMV (7 dpi, n = 11; 10 dpi, n = 12). At 7 and 10 dpi, blood was collected for analysis of creatine kinase activity in the serum. Graphs represent the arithmetic mean ± SEM.