Production of IFNa is an important component of the pDCderived antiviral response. To assess this component of the pDC anti-rotaviral response, the frequency of cells expressing IFNa was determined by flow cytometry. Intracellular IFNa was detected within 4hpi of RRV stimulus, peaked at 6hpi and was sustained until at least 12hpi ( Figure 3A and 3B). IFNa was detected intracellularly at similar frequencies in pDCs exposed to iRRV (which was both infectivity and transcriptionally negative) (p = 0.815; Mann-Whitney) ( Figure 3A and data not shown), suggesting that replication or transcription of rotavirus is largely dispensable for IFNa induction in human pDCs. Intracellular IFNa was abrogated by the addition of RRV neutralizing VP7 or VP4 monoclonal antibodies (either mAb 159 or 2G4), indicating that the observed induction was virus-specific and likely dependent on an event occurring after virus binding and entry ( Figure 3C ) [49] . To identify and quantify IFNa secretion, and to detect other cytokines and chemokines secreted in response to RRV exposure, Luminex assays or ELISAs were performed on supernatants collected from inoculated pDCs at various times post infection. Only supernatants from pDC cultures $85% pure (mean: 90.84%60.75; n = 3-18) were analyzed to minimize the contribution of contaminating cells to the cytokine milieu. A significant IFNa response, approaching 10,000 pg IFNa/ml, was detectable 6h following exposure to live or inactivated RRV ( Figure 4A ). In addition, significant quantities of IFNb, TNFa, interleukin (IL)6, IL8, CXCL10 (IP10), CCL3 (MIP1a), CCL4 (MIP1b), CCL5 (RANTES) and TNFb were detected at various times post infection ( Figure 4B -I and data not shown). Together, these data suggest that live or inactivated RRV rapidly induce production of significant levels of IFNa and other proinflammatory cytokines and chemokines by pDCs.