Vero E6 Produce IFNl Figure 4. Innate responses to SNV in Huh7 and A549 are specifically due to Vero E6-derived IFNl. Total cellular RNA was isolated from Huh7 or A549 cells and used for ISG quantitation by qRT-PCR. (A) Huh7 were treated with SNV (50 mL of virus) or the indicated amount of recombinant human IL29 or IL28A for 3 h prior to MxA mRNA quantitation by qRT-PCR. (B) Huh7 were infected with SNV at an MOI = 1 (50 mL) with or without prior incubation with anti-IL29 or anti-IL28A antibodies at the indicated final concentrations for 1 h. Three hours post infection, ISG56 and MxA mRNA expression was measured by qRT-PCR. (C) SNV or IFNb was preincubated with the indicated amount of anti-IFNb antibody for 1 h. Virus or IFNb was then added to Huh7 cells for 3 h and ISG56 and MxA was measured 3 h post exposure by qRT-PCR. (D) A549 were infected with SNV at an MOI = 1, with or without prior incubation with anti-IL29 blocking antibodies (10 mg/mL). Three hours post infection ISG56 and MxA levels were measured by qRT-PCR. Real-time qRT-PCR results for all experiments (parts A–D) are reported as the mean 6 SEM from triplicate biological experiments. doi:10.1371/journal.pone.0011159.g004 Discussion Herein, we report that Vero E6 cells can recognize viruses and respond by secreting IFNl. The Vero E6 cell line lacks the ability to transcribe type I IFNs as they possess a genetic deletion that ablates the entire type I IFN gene complex on both chromosomes [9]. In addition, it has recently been shown that these cells mount a weak initial IRF3-dependent response to various ligands, which partially accounts for their overall weak innate response to viruses and causes them to be extraordinarily competent for propagating several virus types to high titers [10]. Type III IFNs are transcribed independently of type I IFNs, and their activities are conferred through a different heterodimeric receptor, although both IFNs activate Jak/STAT signaling pathways and therefore their expression results in the transcription of a similar subset of ISGs. The quantity of IFNl thus produced is sufficient to have a dramatic biological effect upon human cells. Different hantaviruses elicit different levels of expression of IFNl, although all of the viruses that we tested induced the secretion of biologically active IFNl in Vero E6 cells. Hantaviruses primarily infect endothelial cells of the microvas- culature and recent advances in the understanding of the innate immune response have prompted laboratories to study early events in viral recognition and host gene expression. Because primary cells are difficult to manipulate, several labs, including ours, have turned to established cell lines for use as a more tractable system for studying viral recognition and innate immune responses to infection. Many of these cell lines are of epithelial origin and express receptors for IFNl. As a consequence, IFNl secreted by Vero E6 cells used to generate viral stocks can cross species and dramatically alter innate gene expression. Herein, we demonstrate that this is true for both the Huh7 and A549 cell lines, which are the two cell lines most commonly used to study the interactions between hantaviruses and human host-cells in in vitro systems. We also confirm herein that innate immune studies performed using cells of endothelial origin are largely unaffected by IFNl carryover from Vero E6 cells. These observations are in accordance with our previous studies showing that endothelial cell responses to hantaviruses vary according to viral serotype and rely on viral entry, in contrast to our observations regarding Huh7 cells [33,34]. An unexpected observation was that Huh7 as well as A549 cells were completely unresponsive to SNV (as well as PHV and ANDV PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11159