Figure 1. SNV stocks induce innate responses in Huh7 independent of titer, replication, and intact virus particles. (A) Huh7 cells were infected with SNV at an MOI = 1 for 1 h. At the time points indicated, cellular mRNA was isolated and used to quantitate viral S-segment RNA (right y-axis) and ISG expression (left y-axis) using qRT- PCR. (B) Vero E6 cells grown in 75 cm2 tissue culture flasks were inoculated with SNV (10 ffu) for 1 h and supernatants were collected at the time points indicated on the x-axis. Virus was then titrated on fresh Vero E6 cells (left y-axis) or used to infect Huh7 cells at equal volumes. ISG56 and MxA expression was measured by qRT-PCR 3 hpi (right y-axis). (C) SNV from 20 dpi Vero E6 stocks was applied to a 100-kDa filter and centrifuged for 10 min. The retentate and filtrate was resuspended to the loading volume and applied to Huh7 cells for 3 h. MxA expression was then measured using by qRT-PCR of total cellular RNA and compared to untreated control (Con) cells. All real-time qRT-PCR results are reported as the mean 6 SEM from triplicate biological experiments. doi:10.1371/journal.pone.0011159.g001 IFNs. We performed western blots using the SNV stocks generated 8 dpi and 20 dpi in Vero E6 cells and concentrated on a 3-kDa filter, after first removing viral particles using a 100-kDa filter. As a positive control, we spiked Vero E6-conditioned medium with Vero E6 Produce IFNl Figure 2. Vero E6-derived SNV stocks induce innate responses via a Jak/STAT pathway and contain IFNl. (A) Hec 1b Jak/STAT knockout cells were treated with poly (I:C) (50 mg/mL) or the indicated stock of virus from Figure 1 at an MOI = 1 for 3 h. Total cellular RNA was isolated and used for qRT-PCR for ISG56 and MxA. Real-time qRT-PCR results are reported as the mean 6 SEM from triplicate biological experiments. (B) Medium supplemented with 50 mg/mL of rhIL29, and SNV derived from Vero E6 8 dpi and 20 dpi were applied to a 100-kDa filter and centrifuged for 10 min. The filtrate was then applied to a 3- kDa filter and centrifuged for 30 min. Equal volumes of the retentate were then used for SDS-PAGE. Western blotting was then performed using an anti-IL29 antibody. doi:10.1371/journal.pone.0011159.g002 recombinant human IL29 (rhIL29) prior to concentration. We were able to detect a band of approximately 32 kDa, correspond- ing to the known size of IL29 (and the rhIL29 used) in the 20 dpi sample, as well as the positive control, but not in Vero E6- conditioned medium alone or in the 8 dpi sample (Figure 2B), which is consistent with their ISG-inducing potential (Figure 1B). To test whether the measured titer of SNV from Vero cells is falsely lowered by the presence of IFNl in viral stocks, we conducted endpoint titers of virus in a series of twofold dilutions to determine whether the titers predicted by the more highly-diluted virus stocks appeared higher than those less diluted, on the assumption that an inhibitor such as IFNl would become diluted and less and less effective in blocking focus production at higher dilutions. We observed that there was no such augmentation in apparent titer at higher dilution, indicating that routinely- measured titers are likely not affected by the presence of IFNl (data not shown). Divergent hantaviruses induce IFNl secretion in Vero E6 cells We asked whether IFNl secretion by Vero E6 cells is specifically induced by SNV, or if IFNl expression is a general feature of infection by hantaviruses. We compared the cellular IFNl response to Prospect Hill virus (PHV), a New World hantavirus thought to be non-pathogenic, to SNV and Andes virus (ANDV), the primary etiologic agents of HCPS in the New World. Vero E6 monolayers were infected for equal amounts of hantavirus (16103 ffu) and supernatants were collected at the indicated time points and used for the detection of IFNl by ELISA or for virus infection by western blotting of nucleocapsid (N). PHV PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11159