Endosomal acidification is required for initiation of the pDC IFNa response to rotavirus To begin to characterize the mechanism by which pDC recognition and subsequent induction of the IFNa response occurs following rotavirus exposure, pDCs were treated with concanamycin A, a potent inhibitor of endosomal acidification, and thus TLR7/9 function [9, 51, 52] , prior to inoculation with either live or inactivated RRV at an moi of 10 ( Figure 8A-8C) . Subsequent examination of intracellular IFNa production revealed significant inhibition in concanamycin-treated pDCs compared to untreated cells (mean 6 SEM, RRV: 83.3%64.6; iRRV: 83.2%64.5; p = 0.01; Mann-Whitney). To determine whether the observed lack of IFNa production was due to a defect in viral entry, concanamycin-treated pDCs were examined for NSP2 expression. Interestingly, NSP2 expression was significantly increased in concanamycin-treated pDCs compared to untreated cells ( Figure 8D and 8E) (mean fold increase: 5.9; p = 0.03; Wilcoxon signed rank test; n = 6). These data indicate that concanamycininduced inhibition of endosomal acidification does not inhibit viral infection of pDCs and hence, presumably, does not restrict viral entry. These data support the conclusion that concanamycin A treatment prevents the pDC IFNa response at the level of ligand recognition, implicating TLR7 or TLR9. Additionally, the data suggest that the observed low levels of viral replication in pDCs ( Figure 1D ) are due, at least in part, to suppression by the pDC IFN response, as NSP2 expression significantly increases following IFNa inhibition ( Figure 8C and 8D) . Notably, these experiments demonstrate that endosomal acidification is required for the pDC IFNa response to rotavirus, and provide indirect evidence for the involvement of an endosomal receptor, such as TLR7 or TLR9, in the induction of this response.