Peak titers of reovirus in the brains of intracranially-inoculated wild-type mice were ,1000-fold higher than those in perorallyinoculated wild-type animals (compare Figures 5E and 6B) . We thought it possible that this difference in viral load might contribute to the dramatic difference in the requirement for Bid in the pathogenesis of reovirus-induced CNS disease following PO and IC inoculation. To test this hypothesis, we inoculated wild-type and Bid-deficient animals intracranially with a considerably lower but still lethal dose of T3SA+, 5 PFU, and monitored infected animals for signs of reovirus encephalitis ( Figure 6C ). In comparison to wild-type mice in which ,95% succumbed to disease, ,70% of Bid-deficient mice developed lethal encephalitis. Moreover, the median survival time of wildtype mice infected with T3SA+ was significantly less (13 days) than that of Bid-deficient mice (15 days). To determine whether this difference in survival correlates with the efficiency of reovirus replication in the CNS, we compared titers of reovirus in brains resected from infected mice at 4, 8, and 12 d post-inoculation ( Figure 6D ). Titers of reovirus in brains of wild-type mice were substantially higher (,10-to 100-fold) at each interval in Results are expressed as the mean ratio of caspase-3/7 activity from infected cell lysates to that from mock-infected cell lysates for triplicate samples. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (B) Wild-type or TRAIL-R-deficient MEFs were adsorbed with T3D at the MOIs shown. After incubation at 37uC for 48 h, cells were stained with AO. Results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. Error bars indicate SD. *, P,0.05 as determined by Student's t-test relative to wild-type MEFs infected at an equivalent MOI. (C) Wild-type and TRAIL-R-deficient MEFs were adsorbed with T3D at an MOI of 2 PFU/cell. The inoculum was removed, and cells were incubated at 37uC for 0, 12, 24 and 48 h. Viral titers were determined after two cycles of freeze-thaw by plaque assay using L929 cells. Results are presented as mean titers from three independent experiments. Error bars indicate SD. (D) Wild-type and TRAIL-R-deficient MEFs were mock-infected or infected with T3D at an MOI of 100 PFU/cell. Whole cell lysates were prepared at 48 h after infection and resolved in 18% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with polyclonal antisera specific for Bid or actin and appropriate HRP-conjugated secondary antibodies and visualized using chemiluminescence. doi:10.1371/journal.ppat.1000980.g004 Figure 5 . Bid modulates reovirus replication and pathogenesis following PO inoculation. (A) Two-day-old wild-type and Bid-deficient mice were inoculated perorally with 10 4 PFU of T3SA+. Mice (n = 23 to 27) were monitored for survival for 21 days. ***, P,0.005 as determined by logrank test in comparison to wild-type mice. (B-E) Two-day-old wild-type and Bid-deficient mice were inoculated perorally with 10 4 PFU of T3SA+ and euthanized at the times shown. Intestines (B), liver (C), heart (D), and brain (E) were resected and homogenized by freeze-thaw and sonication. Viral titers in organ homogenates were determined by plaque assay. Results are expressed as viral titer in organs of single infected animals as indicated by comparison to those in Bid-deficient mice, with those at 4 and 12 d post-inoculation reaching statistical significance. These findings indicate that at a lower viral inoculum, Bid promotes efficient replication of reovirus in the CNS. Collectively, these data suggest that Bid influences reovirus virulence by regulating the growth of reovirus in the brain.