To further investigate the impairment of the IFNa response in pDCs undergoing rotavirus replication, phosphorylation of IRF7, the key transcription factor for the IFNa response in pDCs [50] , was assessed by phosflow ( Figure 5D and 5E). In accordance with the decreased production of IFNa by NSP2 bright cells compared to that of NSP2 dim pDCs, as well as previous findings of IRF7 degradation in rotavirus-infected cells [45, 46] , phosphorylated IRF7 (pIRF7) was substantially decreased in pDCs bright for NSP2 (p = 0.03 versus NSP2 dim pDCs; Wilcoxon signed rank test) ( Figure 5D and 5E), and is similar to that of IFNa -NSP2 -pDCs (p = 0.56; Wilcoxon signed rank test) ( Figure 5E ). To determine whether this decrease in pIRF7 was due to decreased levels of total IRF7 in pDCs, the MFI of total IRF7 was assessed ( Figure 5F ). While the total IRF7 MFI was slightly depressed in NSP2 bright compared to NSP2 dim pDCs, this decrease was not significant (p = 0.44; Wilcoxon signed rank test) and thus does not account for the observed differences in IRF7 phosphorylation. The increased levels of pIRF7 in NSP2 dim pDCs may represent continual, direct stimulus of the IRF7 pathway in virus-positive pDCs, as well as a mechanism by which the IFNa response is directly initiated. Alternately, pIRF7 may be sequestered in a nontraditional cellular compartment prior to degradation, leading to increased protein accumulation. Interestingly, levels of IRF7 phosphorylation in IFNa + NSP2 -pDCs were intermediate to that of NSP2 dim (p = 0.31; Wilcoxon signed rank test) and NSP2 bright or NSP2 -IFNa -pDCs (p = 0.0313; Wilcoxon signed rank test) ( Figure 5E ). The slight depression of pIRF7 in the IFNa + population compared to that observed in NSP2 dim pDCs may be attributable to the enhanced IFNa production in this subpopulation. As pIRF7 is degraded following the initiation of IFNa transcription, cells in which IFNa has been transcribed would be expected to have lower levels of pIRF7 than those negative for IFNa. Notably, inhibition of IFNa secretion, achieved by simultaneous addition of brefeldin A with viral inoculation, decreased the percent of IFNa + pDCs by an average of 63.5% (n = 6, data not shown). This supports the notion that recognition of secreted IFNa is an essential component of the IFNa response following rotavirus exposure, as has been reported with Newcastle disease virus [14] . Importantly, this is the first demonstration of impaired IRF phosphorylation in primary human cells infected with rotavirus.