Figure 7. The pathogenesis of influenza-induced myopathies is likely not direct infection of skeletal muscle in vivo as viral copies are not seen in the gastrocnemius muscle. Young and aged C57BL/6 mice were intranasally infected with 500 EID50 of PR8 influenza. (A) On day 0, 3, 7, 11, and 15 whole gastrocnemius was harvested and RNA was isolated. Total number of copies of influenza PA was determined via RT-qPCR with a positive control used (infected mice lung tissue). Uninfected young and aged murine myoblasts were incubated with 0, 10, or 100 EID50 PR8 influenza for one hour and then were cultured in growth media. Myoblast supernatant was analyzed for chemokine/cytokines via multiplex assay. Detectable cytokines were analyzed by 3-way ANOVA (age x infection condition x time point). While a significant age effect was observed (p<0.05), there was no effect of infection and no interaction of infection and time for myoblast secretion of IL6 (C), CXCL10 (D), CXCL1 (E), and CCL2 (F). At 96 hr post infection, total RNA from the myoblast culture was extracted and total number of copies of influenza PA was determined via RT-qPCR. No viral copies were present in in vitro myoblast cultures (B).