Figure 5. Endothelial cells specifically respond to SNV and are refractory to IFNl. HUVEC were treated with SNV at an MOI = 1 (50 mL of stock virus), rhIL29 (50 ng/mL), rhIL28A (50 ng/mL), or left untreated (con) with or without pre-incubation with the indicated neutralizing antibody for 1 h. Cellular RNA was used to quantitate ISG expression 3 h post exposure. Real-time qRT-PCR results are reported as the mean 6 SEM from triplicate biological experiments. doi:10.1371/journal.pone.0011159.g005 at early time points, data not shown) in that the viruses failed to elicit ISG activation. Blockage of signaling by Vero-derived IFNl using antibodies that neutralize type III IFN completely abrogated the ISG response to virus stocks. Furthermore, despite continuous and high level of SNV viral replication, Huh7 cells never respond by upregulation of ISG56 or MxA, which are markers of activation of the IRF3 and Jak/STAT pathways respectively. These observations are in contrast to a recent report that Huh7 and A549 cells respond to hantaviruses by up regulating ISGs [3]. In the aforementioned study, both Huh7 and A549 cells mounted a strong and early MxA response when exposed PHV as compared to Hantaan virus (HTNV). We observe that stocks of PHV (12dpi) contain high levels of IFNl, whereas the SNV and ANDV stocks contain less IFNl for a given time point. It is possible that in their system, undetected Vero E6-derrived IFNl induced by PHV and contained within virus preparations might account for their finding that PHV induces a robust and early innate response when compared to HTNV. We observe levels of IFN lambda in hantavirus preparations that are sufficient to activate an ISG response when presented to epithelioid cells at a volume corresponding to less than an MOI = 1. This MOI represents approximately 50uL of SNV stock when used to infect near-confluent cells grown in 24-well plate format, and a greater volume of virus stock for both ANDV and PHV, as these viruses typically grow to a lower titer. Interestingly, we observe that this IFNl is highly stable, especially compared to the type I IFNs. We were able to detect high levels of IFNl by ELISA following 5 megarads of gamma irradiation (Fig. 3). We also observe little or no decline in the biological activity of IFNl following UV-inactivation of virus preparations, one freeze-thaw cycle, or storage at 4C for up to one month (data not shown). The lack of innate immune responsiveness to hantaviruses, independent of IFNl, by Huh7 cells suggests that the PRR responsible for recognition of these hantaviruses, at least for SNV, is not RIG-I or Mda5, confirming our previous observations [34]. Huh7 cells, along with A549 cells possess intact RNA helicase pathways, but are unable to mount a TLR3-mediated response to several high titer replicating viruses [36,37]. It is unlikely that this unresponsiveness presented herein is attributed to a virus-encoded interferon antagonist that is efficient enough to completely Vero E6 Produce IFNl suppress ISG transcription, as this is not the observation when using endothelial cells. In support of our evidence that RIG-I is not the PRR used to detect hantaviruses, recent work has shown that RIG-I recognizes RNAs with 59 triphosphate groups [40]. In this study, HTNV was shown to be sensitive to a 59 monophosphate- specific exonuclease, suggesting a post-transcriptional modification of the RNA might be a strategy used by hantaviruses to avoid recognition by RIG-I [40]. Our data also suggests that Vero E6 cells possess an intact hantavirus-sensing PRR and IRF3 pathway. The delayed and prolonged IFNl activation that we observe is likely due to the recently characterized weak endogenous Vero E6 IRF3 transcription factor as well as an impaired positive feedback amplification of the innate response due to the lack of type I IFNs [10]. These inherent characteristics, taken together with their ability to mount a type III IFN response, might lend Vero E6 cells particularly useful for examining the signaling events of early innate responses, and IFN antagonism, by hantaviruses, as well as other virus families. Our finding that PHV can induce a stronger and earlier IFNl response in Vero E6 cells is not surprising. HCPS-causing hantaviruses have been shown to induce a weak IFN response, whereas PHV induces IFNs much more readily [29]. This is likely due to an antagonistic capability of SNV and ANDV upstream of IFN activation. The isolate of ANDV we are using consistently replicates to lower titers than does the SN77734 isolate of SNV, although here we show that both viruses activate a similar IFNl profile. This is possibly due to individual differences in their antagonistic potential in conjunction with their replicative fitness. Conversely, the profile of IFNl induced by PHV in Vero E6 cells reinforces the observations that IFN antagonism by this virus is less efficient. The modulation of IFNl by hantaviruses may have clinically relevant implications. Interestingly, IFNl expression has been observed to be important for human hantavirus infection in vivo. Patients infected with Puumala virus and presenting with nephropathia epidemica (NE) had significantly lower serum levels of IFNl during acute infection compared with healthy control patients. In vitro, HTNV virus replication is inhibited in cells pretreated with IFNl, although post infection treatment does not inhibit replication [38]. Furthermore, it has recently been shown that dendritic cells treated with IFNl, but not type I IFNs, are able to polarize T cells to a Foxp3+ expressing tolerogenic phenotype, indicating that IFNl might skew the immune system toward an anti-inflammatory response [41]. The natural host and reservoir for SNV is the deer mouse, which becomes persistently infected and displays little or no pathology [42]. CD4+ T cells from persistently infected deer mice display a Foxp3+ phenotype, indicative of T regulatory cells, and this may be a mechanism in which the deer mouse avoids an inflammatory-mediated patho- genic state [43]. The mechanism of the T cell regulation is currently unknown, but IFNl might play a role in generating the observed suppressive response. The link between cell culture production of IFNl and in vivo IFNl expression, and its importance in the pathogenic process of hantavirus infection will need to be investigated further. The activation of IFNl by Vero E6 cells demonstrates that these cells mount an innate immune response to viral infection. The IFNl response in Vero E6 is likely delayed because of their inherently weak IRF3 response and lack of type I IFN genes. This ISG response almost certainly affects the quality and quantity of viral stocks generated in this system. The onset of IFNl production in response to hantaviruses correlates with a decrease in viral titers for all viruses tested, suggesting that these cells enter an anti-viral state, albeit inefficiently. Disruption of this innate PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11159