We verified the putative increases in secreted cytokines and chemokines at the protein level by ELISA in 8-14 additional donors. As shown in Figure 2 , PR/8 infection significantly increased secretion of cytokines of TNF-a, IL-6, IFN-a, and IL-29, and CXC chemokines CXCL8-11 as well as CC chemokines CCL2, 4 and 5. Consistent with the mRNA data, CXCL10, CXCL11 and CCL5 were the main chemokines induced by the virus, and AM secreted slightly more IFN-a than IL-29 ( Figure 2 ). Secretion of cytokines and chemokines occurs without the release of a significant amount of infectious viral particles From our previous studies we knew that AM do not release a significant amount of infectious virus particles after infection with human influenza viruses [3, 19] . To investigate whether the infected macrophages were synthesizing viral proteins, we performed a time-course infection experiment in AM from an additional 4 donors using both live ( Figure 3A -D) and UVinactivated PR/8 viruses ( Figure 3E ), examined the kinetics of viral antigen synthesis by staining hemagglutinin (HA) or nucleoprotein, and measured secretion of selected cytokines by ELISA. As shown in Figure 3F , there was a slight increase in viral production at 6 hpi, when about 20% of the cells expressed viral antigens and then no more net increase in viral release as up to 80% of the cells expressed viral proteins by 48 hpi. The viral antigen staining was due to viral replication, since there was no signal with UV-inactivated virus ( Figure 3E ). Despite the abortive release of infectious virus, PR/8 infection induced a time dependent cytokine and chemokine response in human AM ( Figure 3G -K). Viruses triggered an early and rapid secretion of IFN-a and CXCL10 at 6 hpi. Secretion of CCL5 and CXCL8 followed the pattern of the viral protein synthesis increasing with time. The virus-induced increase of TNF-a peaked at 24 hpi and then declined. UV-inactivation abolished the virus-stimulated TNF-a production, significantly decreased secretion of IFN-a, CXCL8, and CCL5. However, the inactive virus was able to stimulate a strong CXCL10 response, although the degree was slightly smaller than that from live PR/8 ( Figure 3I ). The different patterns of the induction suggest that the cytokine response may involve different regulatory mechanisms. In addition, we compared the alterations in mRNA levels of selected innate immune response genes at 3 and 24 hpi for both UV-inactivated PR/8 and live PR/8 infections. Consistent with the protein data, both live and UV-inactivated PR/8 stimulated a large increase in CXCL10 mRNA at both time points. UV-inactivated PR/8 stimulated an up to 4 fold increase of CCL5 and IFNA1. UV-inactivated virus did not alter mRNA levels of RIG-I, TLR7, or ISG56 at either time point (data not shown). These results indicate that viral replication is required for most selected innate immune responses but not required for the CXCL10 response.