llowing the manufacturer’s instructions. To remove any traces of contaminating DNA, extracted RNA was treated with 1 U of RQ1 RNase-free DNase PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19770275 for 1 h at 37C and DNase was heat inactivated 10 min at 65C. Conventional RT-PCR reactions to detect HAstV RNA, IFN-, GAPDH, and ISG56 mRNAs were performed using Expand Reverse Transcriptase and Expand High Fidelity PCR System. HAstV genome amplification was performed using primers A1 and A2 as described previously. IFN-, 3 / 18 HAstV Delays Interferon Induction GAPDH, and ISG56 mRNA amplification was performed using primers described elsewhere. Quantitative real-time RT-PCR assays were carried out using a Mx3000P instrument. Primers and probes for HAstV genome quantification target ORF1b region and have been previously described. Standard curves for HAstV RNA quantification were included in every assay and were generated by using pAVI6 plasmid, which contains the complete genome of HAstV-1 . For quantification of IFN- and GAPDH mRNAs, primers and probes were designed using the Real-Time PCR Assay Design Tool from Integrated DNA Technologies website. IFN- specific primers and probe were 5′ AAG GCC AAG GAG TAC AGT-3′, 5′- CAC AGG CTA GGA GAT CTT CA-3′, and 56-FAM/TAG GAT TTC CAC TCT GAC TAT GGT CCA GG/3IABlk_FQ. GAPDH specific primers and probe were 5′- ACA TCG CTC AGA CAC CAT-3′, 5′- GGG TCA TTG ATG GCA ACA-3′, and 56-FAM/ACC AAA TCC GTT GAC TCC GAC CTT /3IABlk_FQ. For IFN- and GAPDH quantification, a standard curve was constructed in every assay using 5 10-fold serial dilutions of the RNA sample corresponding to the positive and negative controls of the experiment, respectively. GAPDH mRNA titers were used as an endogenous control to normalize all samples versus the number of cells. All samples were quantified at least in duplicate. Reactions were performed using the One step RT qPCR MasterMix Plus kit. The thermal protocol consisted of 30 min at 48C, followed by 10 min at 95C and 40 cycles of 15 s at 95C and 1 min at 60C. Immunofluorescence assays Virus-infected CaCo-2 cells grown on glass coverslips in 24-well plates were rinsed twice with phosphate buffered saline and stained by immunofluorescence as previously described. Samples were analyzed under a fluorescence microscope. Mean percentage of infected cells was calculated from 5 fields of each coverslip, after PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768500 analyzing 
images using ImageJ software. Measurement of antiviral activity by a virus infectivity reduction bioassay The amount of antiviral activity secreted by cells was estimated by using a bioassay in HeLa cells. Before performing the assay, supernatants of HAstV-infected or transfected cells were UV-inactivated. HeLa cells grown on 96-well plates were pretreated with 2-fold dilutions of inactivated supernatant samples for 24 h at 37C, before infection with EMCV at a MOI of 1 TCID50/cell. The development of EMCV-induced cytopathic effect was monitored at 48 h post-infection. Cells pretreated with 2-fold dilutions of culture media containing 1,000 U/ml of recombinant type I IFN were used as a PP-242 chemical information reference control, and levels of antiviral activity were expressed relative to it. ELISA assay for quantification of HAstV capsid protein An indirect enzyme-linked immunoassay was performed on serial 4-fold dilutions of samples. Astrovirus antigens in cell lysates were captured with mAb 8E7 and detected by using a rabbit polyclonal anti-HAstV antibody. Specifically bound antibodies were revealed by a peroxidase-conjugated anti-rabbit IgG antibo