se to 100%. Percentages were higher when infecting cells with a MOI of 10, with a maximum of 20.4 5.8% at 48 hpi. At 96 hpi, cell damage due to infection was too strong to allow proper interpretation of IF data. At 12 hpi, IRF3 was localized to the cytoplasm of all HAstV-infected cells. Together these results 5 / 18 HAstV Delays Interferon Induction Fig 1. Induction of an IFN response is delayed during HAstV infection. Temporal analysis of induction of IFN- and ISG56 mRNA expression by in CaCo-2 cells infected with HAstV at a MOI of 1. Mockinfected cells, cells treated for 24 h with exogenous IFN at 1,000 U/ml, and 
polyI:C-transfected cells were used as controls. HAstV growth curve on CaCo-2 cells at 2 different MOIs. Total HAstV RNA was measured by qRT-PCR at the indicated times post-infection. Data represent mean values of duplicate wells and error bars represent the standard error of the mean. doi:10.1371/journal.pone.0123087.g001 indicate that, despite the high number of HAstV-infected cells, the IFN response in infected cultures was attenuated. To determine if transcription of IFN- induced by HAstV could result in production and cell release of type I IFN, we measured the presence of antiviral activity in supernatants from HAstV-infected cells at two different MOIs, using a virus infectivity reduction bioassay, using treatment with 1,000U of type I IFN as a reference control. In order to make sure that no residual HAstVs would remain in the supernatants, samples were CF-101 site inactivated by a 1-h UV incubation prior to the assay. Total viral inactivation was confirmed by lack of positive cells by IF analysis. Antiviral activity against EMCV could be detected in supernatants of HAstV-infected cells starting at 48 hpi, suggesting that activation of IFN- gene results in protein production and secretion to the extracellular environment. IFN response to HAstV is dependent on virus replication To determine if productive viral replication was required for induction of an innate response, CaCo-2 cells were inoculated with infectious HAstV or an equivalent amount of UV-inactivated virus. No differences were observed in the viral dose as confirmed by RT-qPCR. Antiviral activity in the supernatant of cultures was never observed in cells infected with 6 / 18 HAstV Delays Interferon Induction 7 / 18 HAstV Delays Interferon Induction Fig 2. Analysis of the level of IFN response in HAstV-infected cells. Quantification of IFN- mRNA levels by qRT-PCR during infection of CaCo-2 cells at a MOI of 1. qRT-PCR values with primers specific for human IFN- mRNA were normalized to endogenous GAPDH mRNA levels at each time point, and results were expressed as fold induction of IFN- expression versus 0 hpi. PolyI:C-transfected cells at 24 hpt were used as a PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19767819 positive control. Results shown are the mean values of 2 independent experiments and error bars represent the SEM. Kinetic analysis of IRF3 subcellular localization during HAstV infection at a MOI of 1. PolyI:C transfected cells fixed at 24 h post-transfection and mock-infected cells fixed at 48 and 96 hpi were used as positive and negative controls, respectively. Cells were labeled for HAstV capsid protein and IRF3. White arrows indicate cells with nuclear PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768759 translocation of IRF3. Percentage of cells with translocation of IRF3 into the nucleus. Data were calculated after counting the number of cells with nuclear IRF3 from 5 fields from coverslips from 23 independent experiments using the Image J software. doi:10.137