Fig. 5.
Development of a hybridization-based reovirus T3D viral population diversity assay. (A) Sequence alignment showing groups of silent point mutations introduced into the reovirus T3D M1 gene segment. The amino acid sequence is shown at the top. (B) Blots demonstrating specificity of each probe for its cognate viral RT-PCR product. (C) Serial passage competition experiment showing maintenance of all pool members. L929 cells were adsorbed with equivalent PFU of each reovirus pool member. Infected cells were collected at 24 hpi and used to initiate another cycle of replication in naïve cells. Following seven passages, the ratios of viruses were compared by hybridization assay. (D) Representative blot of RT-PCR products from a reovirus-infected IFNAR−/− mouse following intramuscular inoculation. Mice were inoculated intramuscularly with 107 PFU total of the nine genetically marked reoviruses. Tissues were collected at 72 hpi, and viral population diversity was assessed using the hybridization assay. (E) Relative prevalence of each reovirus pool member in all mouse tissues collected during this study. Data were derived from tissues of 63 mice. The relatively equal prevalence of each pool member indicates that pool members do not have significant fitness differences in vivo.