Although the ability of pDCs to mount an IFNa response to viral infection is well described, the only documented viral TLR ligands for pDCs are ssRNA and DNA [2, 3, 7, 8, 9] . As rotavirus is a dsRNA virus and since psoralen-treated, transcriptionallyinactive rotavirus efficiently induced IFNa (Figures 3 and 4A) , we investigated the viral requirements for IFNa induction. pDCs stimulated with equal quantities (see Methods describing particle quantification) of purified TLPs, DLPs, empty particles, or genome-free recombinant virus-like particles (VLPs) were stained for intracellular IFNa. As expected, density gradient purified RRV TLPs stimulated similar levels (p = 0.695; Wilcoxon signed rank test; n = 10) of IFNa production compared to pDCs treated with unpurified live or inactivated RRV expressing equivalent amounts of VP4, as measured by hemagglutination titer (Figure 7 ). pDCs exposed to 2/6 genome-containing RRV DLPs expressed neither IFNa nor NSP2, indicating a likely dependence on VP4 or VP7 for viral entry, as previously reported [32] . Alternately, one or both of these viral proteins could be serving as the pathogenassociated molecular pattern (PAMP) responsible for inducing the pDC IFNa response. To address this, pDCs were stimulated with 2/4/6/7 VLPs or 2/6/7 green fluorescent protein (GFP)-VLPs. Both 2/6/7 GFP-VLPs and 2/4/6/7 VLPs were capable of entering pDCs, but failed to induce IFNa (Figure 7 and data not shown). 2/6 VLPs were comparatively defective in the ability to enter pDCs (data not shown), thus supporting the hypothesis that DLPs fail to induce IFNa due to inefficient viral entry. The ability of infectious SA11, the parental virus from which the 2/4/6/7 VLPs were derived, to induce IFNa production ( Figure 7) suggests that the lack of IFNa following inoculation with 2/4/6/7 VLPs was due to the absence of a viral genome, and not to strain variation in surface proteins. Consistent with this hypothesis, IFNa was detected in very few pDCs exposed to empty TLPs, as compared to pDCs exposed to equal quantities of genomecontaining TLPs. Instead, IFNa production by pDCs exposed to empty particles was consistent with that observed in pDCs inoculated with an equal infectious dose of TLPs, suggesting that residual contaminating viral genome was responsible for IFNa production by the empty TLPs. Together, these data indicate that viral entry, mediated by VP4 and/or VP7, and the viral dsRNA genome are likely required for rotavirus-induced IFNa production by pDCs.