response may render these cell types more efficient vessels for propagating virus stocks. This work has implications for all systems utilizing Vero E6-derived virus preparations and furthers the field of innate immunity to hantaviruses. Materials and Methods Cells, viruses and reagents We purchased human hepatoma cells (Huh7) from Apath (New York). A549 and Vero E6 cells were purchased from ATCC. We grew cells in DMEM (Invitrogen) containing 10% FCS, 1X essential amino acids (Gibco), pen/strep, and gentamicin. We purchased pooled primary human umbilical vein endothelial cells (HUVEC), from Clonetics (San Diego, CA) and propagated them in EBM-2 medium supplemented with the Clonetics bullet kit, which supplies growth factors including vascular endothelial growth factor (VEGF). We previously described the isolation of Sin Nombre virus (SNV) strain SN77734 from a New Mexico deer mouse [42]. We obtained Andes virus (ANDV) strain CHI-7931 from H. Galeno, Instituto Salud Publico, Santiago, Chile and Prospect Hill virus (PHV) strain PH-1 from R. Yanagihara (Univ. of Hawaii). Hantaviruses were propagated and titrated on Vero E6 cells (ATCC) under strict standard operating procedures using biosafety level 3 (BSL3) facilities and practices (CDC registration number C20041018-0267). For virus infections, we seeded cells in 48- or 24-well plates at a density to achieve 90–95% confluence after overnight incubation at 37uC in 5% CO2. Treatment was done essentially as described previously [33]. For antibody blocking studies, we incubated viruses or recombinant proteins with the indicated concentrations of anti- IL29, anti-IL28A, or anti-IFNb (R&D Systems, AF1598, AF1587 or AF814, respectively) for 0.5 h at room temp. The resulting mixture was then added to cells and incubated for 3 h prior to performing the indicated assays. Recombinant proteins used included rhIL29, rhIL28A and rhIFNb (R&D Systems, 1598-IL- 025, 1587-IL-025 and 11415-1, respectively). For poly (I:C) treatment, we seeded cells as above and at the time of infection, and exposed cells to 10% DMEM containing 50 mg/mL poly (I:C) (Sigma). Virus titrations We infected monolayers of Vero E6 cells grown in 24-well plates with 200 mL of serial 10-fold diluted virus for 2 h. We then removed the inoculum and overlaid the cells with a 1.2% solution of methylcellulose in a final concentration of 1X DMEM, 2.5% FBS. The cells were incubated for seven days prior to staining for foci using a rabbit polyclonal anti-N antibody followed by the addition of a 1:1000 dilution of an HRP-conjugated anti-rabbit- antibody (Jackson ImmunoResearch). Foci were developed using a metal-enhanced DAB substrate kit (Thermo Scientific). Real-Time SYBR Green and TaqMan RT-PCR For SYBR Green RT-PCR, we performed reverse transcription using 2 mg of total RNA extracted from cell cultures using the RNeasy kit (Qiagen) in 40 mL reactions using the Applied Biosystems TaqMan Reverse Transcription reagents kit (ABI, Foster City, CA). For PCR, we used the Applied Biosystems Power SYBR Green reagents kit to perform reactions in triplicate using 3 mL of cDNA and a 0.4 mM final concentration of each primer in Reference Vero E6 Produce IFNl a 25 mL total reaction volume. The primers we used were specific for: b-actin, sense (59-ccatcatgaagtgtgacgtgg-39) and antisense (59- gtccgcctagaagcatttgcg-39); ISG56, sense (59-tctcagaggagcctggctaag- 39) and antisense (59-ccacactgtatttggtgtctagg-39); MxA, sense (59- tgatccagctgctgcatccc-39) and antisense (59-ggcgcaccttctcctcatac-39). We calculated the fold-change for each gene using the mean of the change in CT values (DCT) normalized to the CT values of GAPDH for each sample (22DDCt) [33]. We performed TaqMan RT-PCR for SNV viral S-segment RNA as previously described [33]. We quantified the viral RNA using a standard curve generated using S-segment templates of known copy number. Supernatant filtration We added 1 mL of the Vero E6-derived virus stocks to 100-kDa filtration units (Amicon) and centrifuged them at 40006g for 15 min. The retentates and filtrates were resuspended to a final volume of 1 mL and equal volumes were added to Huh7 cells. For the samples used to detect IL29 by western blotting, we applied the filtrate of 100-kDa filter units to 3-kDa filter units (Amicon) and centrifuged them at 40006g for 45 min. The retentate was then used directly in assays for ISG induction. Western blotting For IL29 detection, we boiled the retentates of the 3-kDa filtration units along with a 4% SDS loading buffer for 5 min. We then separated retentates on a 12.5% SDS-PAGE gel and transferred the proteins to PVDF membranes. Membranes were then probed with a primary polyclonal rabbit anti-IL29 antibody (R&D Systems) overnight at 4C at a concentration of 0.2 mg/mL followed by an incubation with a 1:10,000 dilution of a HRP- labeled anti-rabbit IgG secondary antibody for 1 h (Jackson ImmunoResearch). For hantavirus N detection, we used the methods described previously [34]. ELISA of IL29 We performed ELISA using the human IL29 DuoSet ELISA Development System (R&D Systems, DY1598) as per their recommended protocol, with the exception of coating the plates with a polyclonal anti-human IL29 capture antibody (R&D Systems, AF1598). This antibody was used for its greater crossreactivity to African green monkey-derived IFNl. We added 100 mL of Vero E6-derived viral stocks that had been subjected to 5 megarads of gamma irradiation, or Vero E6-conditioned medium as a control. The concentrations of IL29 were interpreted from a curve generated using 2-fold dilutions of a recombinant IL29 standard starting at 8000 pg/mL. All data were reported as the average of samples and standards run in duplicate wells. Statistical analysis Statistical analyses were performed on replicate samples using an unpaired Student’s t test. The mean 6 standard error of the mean is represented and significance (P,0.05) is reported where appropriate. Author Contributions Conceived and designed the experiments: JP PH MGW HE BH. Performed the experiments: JP PH MAR CY. Analyzed the data: JP PH BH. Contributed reagents/materials/analysis tools: MGW HE HF BH. Wrote the paper: JP HE HF BH. 1. Song JW, Baek LJ, Gavrilovskaya IN, Mackow ER, Hjelle B, et al. (1996) Sequence analysis of the complete S genomic segment of a newly identified hantavirus isolated from the white-footed mouse (Peromyscus leucopus): phylogenetic relationship with other sigmodontine rodent-borne hantaviruses. Virus Genes 12: 249–256. PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11159