induced a robust IFNl response by 12 dpi and both ANDV and SNV induced modest levels of IFNl at late time points post infection (Figure 3A). Mock-infected cultures had low levels of IFNl for all time points, indicating that the increase in IFNl secretion is not a result of prolonged cell culture or cell death at late time points, but is virus-specific. This method for generating hantavirus stocks using Vero E6 cells is typical and the IFNl measured in these virus preparations represents the absolute amount of cytokine contained within a milliliter of a given virus stock. The low level of IL29 detection in the control samples is likely due to our substitution of a polyclonal detection antibody to achieve greater cross reactivity to the African green monkey- derived cytokine. Western blotting showed an accumulation of N protein in the supernatants, confirming that Vero E6 cells were productively infected by all three viruses (Figure 3B). Activation of ISGs by SNV stocks in Huh7 and A549 is due to IFNl Type III IFNs are potent stimulators of the innate immune response. We suspected that the initial ISG response in Huh7 cells exposed to SNV stocks (Figure 1A) is due to Vero E6 cell-derived IFNl. We assayed whether recombinant type III IFNs were able to induce an ISG response in Huh7 similar to that observed by our SNV stocks. Both IL29 (IFNl1) and IL28A (IFNl2) induced a strong, dose-dependent, MxA response in these cells (Figure 4A). We then analyzed the ability of anti-IL29 or anti-IL28A neutralizing antibodies to block ISG responses by pre-incubation with our viral stocks (MOI = 1, corresponding to 50uL of viral stock). Both neutralizing antibodies were able to efficiently inhibit ISG56 and MxA gene inductions in a dose-dependent manner (Figure 4B). Because both anti-IL29 and anti-IL28A antibodies blocked ISG inductions almost completely at the highest concen- trations used (10 mg/mL), we assayed whether these antibodies cross react by blocking recombinant IL28A and IL29 respectively, and found that both antibodies cross reacted well with exogenous IFNl Vero E6 Produce IFNl proteins as measured by their ability to neutralize ISG activation in Huh7 cells (data not shown). As a control for antibody specificity and type III IFN sensitivity, we used recombinant IFNb along with anti-IFNb neutralizing antibodies and observed that although recombinant IFNb induces a robust ISG response, the anti-IFNb neutralizing antibody only inhibited recombinant IFNb-mediated ISG responses, and not inductions by the SNV stock containing IFNl, indicating that the anti-IFN antibodies are neutralizing only their homologous targets (Figure 4C). Along with Huh7, A549 cells are commonly used in hantavirus research and this human lung carcinoma cell line has been used to study innate immune responses in the context of infection by hantavirus [3,35,38,39]. We asked whether this cell type is able to respond to SNV particles themselves, or whether ISG responses in A549 cells are attributed to the IFNl contained within Vero E6-generated stocks. Similar to Huh7 cells, pre-incubation of SNV stocks with anti-IFNl antibodies completely inhibited ISG responses observed in A549 when exposed to SNV particles at an early time point (Figure 4D). HUVEC respond specifically to SNV but not to IFNl Endothelial cells are major target of hantaviruses in vivo and lack the IL28Ra receptor, which renders them incapable of responding to IFNl [14]. We, as well as other groups, have reported that primary endothelial cells respond to hantavirus infection by up- regulating ISGs. We asked whether IFNl contained within our viral stocks might play a role in ISG responses in HUVEC as they do in Huh7 and A549. We found that HUVEC transcribe only very low levels of ISG56 and MxA when exposed to either rhIL29 or rhIL28A. Furthermore, pre-incubation of SNV stocks with IL29 and IL28A neutralizing antibodies at doses shown to block IFNl-mediated ISG induction in Huh7 and A549 cell lines, did not inhibit their ability to respond to SNV (Figure 5). This is consistent with our previous observations that HUVEC responses to SNV are specific, as SNV-specific neutralizing antibodies completely blocks ISG responses to virus [33]. Figure 3. Divergent hantaviruses induce IFNl in Vero E6 cells independent of virus replication. Vero E6 cells were grown in 75 cm2 culture flasks and infected with 103 ffu of PHV, SNV or ANDV for 1 h, or left untreated (Con). Supernatants were collected at the indicated time points post infection and total medium was replaced. (A) Equal volumes of supernatants were used in an ELISA to detect IL29. Error bars represent the standard deviation between duplicate ELISA experiments. (B) Equal volumes of supernatants were used for SDS-PAGE followed by western blotting. Membranes were probed with an anti-hantavirus N antibody. doi:10.1371/journal.pone.0011159.g003 PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11159