FIG. 8.

The absence of IFN-α/β pathway components neither restores EEEV infectivity for DLN cells in vivo nor alleviates the restriction on EEEV productive infection and genome translation in myeloid lineage cells in vitro. (A and B) Mice lacking PKR, RNase L, and Mx (TD); mice lacking IFN-α/β receptor-mediated signaling (IFNAR1^−/−); or normal congenic controls (129/Pas) were inoculated subcutaneously in each rear footpad with equal doses of EREP (black bars in panels A and B), VREP (white bars in panel A), or SBREP (hatched bars in panel B) replicon particles expressing fLUC. Data comparing EREP to VREP (A) and EREP to SBREP (B) are presented in separate panels as these were independent experiments. For panels A and B, DLNs were harvested at 8 h p.i. and fLUC activity was quantified by in vitro luciferase assay. Datum points are presented as RLU per DLN ± standard deviation where n = 8. (C) Primary cDCs generated from 129/Pas (black bars) or IFNAR1^−/− (white bars) mice were infected with EEEV, SB, or VEEV at equal MOIs (0.1). Titers of progeny virus released into the supernatant were determined by plaque assay. Values represent the geometric mean virus titers (log₁₀ PFU/ml) at 48 h p.i. ± standard deviation where n = 3 and confidence limits are indicated. (D) Primary cDCs generated from 129/Pas (black bars), TD (hatched bars), or IFNAR1^−/− (white bars) mice were infected with fLUC-expressing EREP or SBREP conventional replicon particles (MOI, 1). Cells were harvested for luciferase activity assay at 24 h p.i. Values represent the geometric mean fLUC activities (log₁₀ RLU/μg) ± standard deviations where n = 3, and confidence limits are indicated. (E) Primary cDCs derived from 129/Pas (black circles), TD (white circles), or IFNAR1^−/− (black squares) mice were infected with EREP-nsP3-fLUC replicon particles (MOI, 1). Cells were harvested for luciferase activity assay at various times p.i. For panels C to E, values represent the geometric mean fLUC activities (log₁₀ RLU/μg) ± standard deviations where n = 3. A single asterisk in panel E indicates that there was no significant elevation of fLUC activity in the absence of PKR/RNase L or the IFN-α/β receptor versus 129/Pas control cells (P > 0.5).