Microarray identification of host responses to dengue virus replication using the HepG2 infection model Viral replication in HepG2 cells infected with dengue virus TSV01 for 3, 6, 12, 24, 48 and 72 hours, compared to heat inactivated virus, was determined by plaque assay of the virus released in the cell culture medium ( Figure 1A ), FACS analysis of infected cells labeled intracellularly with an Alexa 674-conjugated antibody against dengue E protein (4G2) ( Figure 1B ) and real-time PCR analysis of viral RNA ( Figure 1C ). All three methods showed that new viral replication began after 24 hours and reached a plateau at 72 hrs, with FACS analysis showing 28% of cells infected at this point. After 72 hrs a degree of cell death become apparent (data not shown) and the experiment was not continued. Analysis of microarrays, performed in duplicate (dye-swapped) on three biological replicates at each time point, comparing infectious with heat inactivated virus; using a SAM q value (false discovery threshold) of 25%, revealed no significantly differentially expressed genes at 3, 6, 12 or 24 hrs post infection. However, there were 24 transcripts identified at 48 hrs and 124 at 72 hrs (a total of 132 transcripts representing 124 genes; Table S1 ) that were differentially expressed. At both 48 hrs and 72 hrs, clustering of transcripts using PANTHER analysis identified the IFN-mediated immunity pathway as the most significant with a P value of 10 215 (at 72 hrs). Genes that are typically induced after type I IFN stimulation, including OAS1, OAS2, OAS3, OASL, STAT1, STAT2, MX1, IFIH and IFNb, featured prominently in this cluster ( Figure 2 and Table S1 ). Further analysis of the 124 genes suggested the involvement of NF-kB-mediated cytokine/chemokine responses (NFKBIB, NFKB1A, TNFA1P, CCL4, CCL5, IP-10 and I-TAC amongst others) and ubiquitin related genes (HERC5, HERC6, UBE2L6, USP15 and others) ( Figure 2 ). Although not completely overlapping, a core host response to pathogen involving the IFN response and the NF-kB-mediated immune defense response [10] was observed in our array results. Other genes that were significantly changed included those associated with cell signaling, lipid metabolism, cell cycle and vesicular transport (Table S1 ). We did not detect any significantly down regulated genes in our system. All the microarray data were deposited in a public database accessible at http://www.ncbi.nlm. nih.gov/projects/geo (accession number is GSE6048).