three genes that encode proteins for IFNl1 (IL29), IFNl2 (IL28A) and IFNl3 (IL28B). IFNl expression is controlled by pattern recognition receptor PRR activation, which activates and mobilizes IRF3 and IRF7, which in turn bind to the interferon- stimulated response elements (ISREs) of these genes, along with several other genes. IFNls signal through a heterodimeric cell surface receptor comprised of IFNLR1 and IL10R2 [12,13]. Expression of the IFNLR1 is cell-type specific, with high expression in epithelial cells and little or no expression in endothelial and fibroblast cells, rendering the latter unresponsive to the effects of IFNl [14,15]. Receptor engagement converges with that of the type I IFN receptors in that both activate the Jak/ STAT signaling pathway leading to the formation of ISGF3 (STAT1/STAT2/IRF9), a transcription factor that regulates the expression of several ISGs, including MxA [16,17]. Therefore, both the expression and function of IFNls is much the same as for the type I IFNs. Sin Nombre virus (SNV) and Andes virus (ANDV) are the most important agents of hantavirus cardiopulmonary syndrome (HCPS) in North America and South America, respectively [7,18,19]. HCPS is characterized by pulmonary edema due to capillary leak, followed by cardiogenic shock. Approximately 500 cases by SNV-related viruses have been reported in the U.S. since 1993 with a 32% case-fatality ratio (www.cdc.gov/ncidod/ diseases/hanta/hps/index.htm). These viruses are carried by distinct rodent hosts and transmitted to humans by inhalation of virus-contaminated urine, feces and/or saliva. [20]. Vascular endothelial cells are thought to be the major site of viral replication in humans, and infected cells secrete high levels of chemokines and cytokines as a result [21,22,23,24]. As in many viral systems, it appears that pathogenic hantaviruses possess mechanisms to antagonize the innate immune system and this antagonism has been hypothesized to play a role in the pathogenic process [25,26,27,28]. For example, Prospect Hill virus (PHV), a New World hantavirus which has not been identified as a human pathogen, induces a strong IFN response, whereas the induction by ANDV is much weaker, likely correlating with their IFN antagonism efficiencies and possibly their pathogenic potential [29]. Recent work by our laboratory, as well as others, has focused on the mechanisms of PAMP-PRR engagement and the processes involved in antiviral activities and IFN antagonism [3,29,30,31,32,33,34,35]. Many of these studies have also used human cell lines to characterize these interactions, including Huh7, a hepatoma cell line, as well as A549, a lung carcinoma cell line. Both of these cell lines are deficient in the TLR3 PRR axis but support hantavirus replication [3,34,36,37]. We have previously demonstrated that SNV can induce a strong innate immune response in human umbilical vein endothelial cells (HUVEC) as well as Huh7 cells. We observed several differences in innate immune activation upon infection with hantaviruses. For example, we observed a strong innate response in Huh7 despite blocking virus entry, which was not the observation in HUVEC [33,34]. Therefore, we hypothesized the recognition mechanisms differ between endothelial cells and these cell lines and that a viral surface protein or a soluble mediator of the innate immune response may be responsible for innate immune stimulation. In this study we found that Vero E6 cells mount an innate immune response and secrete IFNl following infection by several hantaviruses, and were able to show that Vero E6-derived IFNl is solely responsible for the induction of ISGs in both Huh7 and A549 cells, whereas ISG stimulation in primary human endothe- lial cells is virus-specific. This is the first report demonstrating that Vero E6 cells secrete IFNl in response to viral infection and this response can in turn elicit downstream biological processes. Vero E6 Produce IFNl Results SNV stock virus generated in Vero E6 cells activates a transient ISG response in Huh7 independent of virus titer, replication or intact particles We have previously demonstrated that, although both primary endothelial cells and the hepatoma cell line Huh7 respond to infection by hantaviruses by up-regulating ISGs, we observed several differences in cellular entry and transcription factor requirements that suggested a divergent PAMP and/or PRR axis had been activated in these distinct cell types [33,34]. Because ISG inductions were observed in Huh7 in response to SNV stocks independent of entry and infection, we hypothesized that the ISG- activating component of the viral stocks may not be associated with infectious virus or the viral particle itself, but may be a soluble component derived from either the virus, or the Vero E6 cells used to propagate the virus. To test our hypothesis, we examined the kinetics of ISG56 and MxA activation by live SNV and compared this to viral replication kinetics in Huh7 cells. SNV induced a strong ISG response upon initial infection, however, this response was transient and almost completely abated by one day post infection (dpi), despite the continuous accumulation of viral S- segment vRNA in Huh7 cells (Figure 1A). We verified that Huh7 cells were productively infected, since we were also able to use their supernatants to infect fresh Vero E6 cells and focus assays performed on Vero E6 cells showed titers of up to 104 ffu/mL (data not shown). These results indicate that either the products of viral replication are not required for ISG stimulation, or that the virus can efficiently antagonize innate immune responses following replication, thus inhibiting ISG activation. We then asked whether the ISG-activating potential in Huh7 is a function of viral titer upon initial infection. For this, we generated a time-course series of SNV stocks in Vero E6 cells and assayed both their viral titer on fresh Vero E6 cells and their ISG-inducing potential on Huh7 cells (Figure 1B). We observed that ISG induction is dissociable from the viral titer, with the highest ISG-inducing activity exhibited by the 20 dpi virus stocks, which had the lowest viral titers. To test whether a soluble mediator of innate immunity is responsible for the observed ISG transcription, we examined whether the ISG- activating fraction is physically dissociable from intact viral particles by filtration. We applied SNV stocks to a 100-kDa cut- off Amicon centrifugation filters and collected the filtrate and retentate, both of which we resuspended to the starting volume by the addition of cell culture medium. We were unable to detect replication-competent virus in the filtrate by titration (data not shown). When these components were applied to Huh7 cells, we observed that the ISG activating fraction was contained within the filtrate, indicating that a soluble component derived from the Vero E6 cell stocks, or viral components of less than 100-kDa relative mass are responsible for ISG inductions (Figure 1C). Vero E6 cells secrete IFNl in response to SNV We screened several human cell lines for their ability to activate ISG56 and MxA in response to SNV. In most cases, adherent human cell lines were able to respond, as assessed by their strong and early induction of expression of these genes. An exception to this was a HEC 1b cell line that is deficient in Jak/STAT signaling (kindly provided by Marc Wathelet). Poly (I:C) (50 mg/mL) was able to induce a strong ISG56 response, but failed to induce MxA, which requires IFN signaling via Jak1/STAT (Figure 2A). SNV stocks failed to induce a response in these cells. Both type I and type III IFNs utilize the Jak1/STAT signaling pathway to exert their effects. Since Vero E6 cells are unable to express type I IFNs, we hypothesized that SNV-infected Vero E6 cells express type III PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11159