Table S1 HepG2 Microarray Gene List. List of HepG2 transcripts (n = 132), identified as differentially expressed by SAM analysis, in response to dengue virus TSV01 compared to heat inactivated virus 48 and 72 hours post infection. Transcripts are placed in groups according to biological processes. qV is the SAM calculated q value for each gene (following SAM significance selection based on a Delta value calculated from the variance between the sample sets, see Material and Methods). F.C. indicates fold change. ''-'' represents no significant change. Genes selected for real-time PCR validation through a TaqMan low density array (TLDA) platform are indicated by a tick. Found at: doi:10.1371/journal.pntd.0000086.s001 (0.28 MB DOC) Table S2 Quantitative PCR by Taqman based low density array in HepG2, A549 and Singapore dengue fever patients. Fold increase in gene expression as determined by quantitative PCR. For HepG2 and A549, the fold change was calculated based on dengue virus infection over heat-inactivated virus infection. For dengue fever patients (Patients), the fold change was calculated using patient blood samples collected at the first visit (,1-2 days after onset of fever) over the convalescence (3-4 weeks after the acute fever). Upregulation is shown in black and down-regulation in red. ''1.0'' represents no significant change (significance determined by q value ,5, see Material and Method). Genes that were significantly up-regulated in at least one time point in HepG2 and in at least one time point in A549 and in Patients are indicated with P-values, calculated by standard student T test and selected based on a cut off at P,0.05. Found at: doi:10.1371/journal.pntd.0000086.s002 (0.19 MB DOC) Species-specific evolution of immune receptor tyrosine based activation motif-containing CEACAM1-related immune receptors in the dog BACKGROUND: Although the impact of pathogens on the evolution of the mammalian immune system is still under debate, proteins, which both regulate immune responses and serve as cellular receptors for pathogens should be at the forefront of pathogen-driven host evolution. The CEA (carcinoembryonic antigen) gene family codes for such proteins and indeed shows tremendous species-specific variation between human and rodents. Since little is known about the CEA gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging CEA family by analyzing the CEA family of the dog. RESULTS: Here we describe the complete CEA gene family in the dog. We found that the gene coding for the ITIM-bearing immunoregulatory molecule CEACAM1 gave rise to a recent expansion of the canine CEA gene family by gene duplication, similar to that previously found in humans and mice. However, while the murine and human CEACAMs (carcinoembryonic antigen-related cell adhesion molecules) are predominantly secreted and GPI-anchored, respectively, in the dog, most of the CEACAMs represent ITAM-bearing transmembrane proteins. One of these proteins, CEACAM28, exhibits nearly complete sequence identity with the ligand-binding N domain of CEACAM1, but antagonizing signaling motifs in the cytoplasmic tail. Comparison of nonsynonymous and synonymous substitutions indicates that the CEACAM28 N domain is under the strongest purifying selection of all canine CEACAM1-related CEACAMs. In addition, CEACAM28 shows a similar expression pattern in resting immune cells and tissues as CEACAM1. However, upon activation CEACAM28 mRNA and CEACAM1 mRNA are differentially regulated. CONCLUSION: Thus, CEACAM1 and CEACAM28 are the first paired immune receptors identified within the CEA gene family, which are expressed on T cells and are most likely involved in the fine-tuning of T cell responses. The direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that CEACAM28 evolved in response to selective pressure imposed by species-specific pathogens. The evolution of immunoglobulin superfamily (IgSF) members is largely influenced by the nature of their ligands. The extracellular part of IgSF members which are predominantly expressed in the nervous system are well conserved, while the extracellular domains of members expressed by immune cells or cells involved in reproduction tend to diversify much more rapidly [1] . The CEA gene family, which belongs to the IgSF, is a tandemly clustered multigene family. Such gene families are subject to rapid evolution due to ongoing gene duplication, deletion and mutational events [2] . At present, the CEA family is subdivided into two main subgroups: the CEA-related cell adhesion molecule (CEACAM) and the pregnancy-specific glycoprotein (PSG) subgroups. The CEACAM subgroup in humans consists of 12 members composed of a single immunoglobulin variable (IgV)-like N-terminal (N) domain followed by zero to six Ig constant (IgC)-like domains of A and B subtypes and one member which consists of two IgC-like domains and two IgV-like domains, one at each end of the molecule. CEACAM1, CEACAM3, CEACAM4, CEACAM18, CEACAM19, CEACAM20 and CEACAM21 are transmembrane molecules while CEA/ CEACAM5, CEACAM6, CEACAM7, and CEACAM8 are linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors and CEACAM16 is most likely a secreted molecule. For CEACAM1 (also known as CD66a, BGP and C-CAM105) and the recently discovered CEACAM16, CEACAM18, CEACAM19 and CEACAM20, but not for other family members have orthologs been identified in rodents [3] . In addition to these orthologs, the transmembrane-bound CEACAM2 (in mice), CEACAM17 (in mice and rats) and the secreted members CEACAM9, CEACAM10, CEACAM11 CEACAM12, CEACAM13, CEACAM14, CEACAM15 (in mice and rats) exist in rodents [3, 4] . In mice, rats and cattle, at least two CEACAM1 alleles each have been identified, which differ considerably in their N domain sequences [5] . No such allelic variation has been observed in humans. The PSG, which are specifically expressed in the trophoblast, are the most abundant fetal proteins in the maternal bloodstream during late human pregnancy. The PSGs are thought to play a pivotal role in the regulation of the maternal immune response to the fetal semi-allograft [6] . The diversity of the CEACAM/PSG family of proteins is further enhanced by species-specific differential splicing of several family members [7] . As expected from the variable structure of the CEA family members, these molecules have diverse functions. It is well established that various CEA family members play crucial roles in cell-cell adhesion, tumor development, angiogenesis, insulin metabolism, reproduction and, as more recently realized, in immunity [8] . CEACAM1 functions as a natural killer cell inhibitory receptor [9, 10] , regulates T and B cell proliferation [11, 12] , induces dendritic cell maturation [13] and facilitates granulocyte and monocyte survival [14, 15] . In addition, CEACAM3, CEACAM6 and CEACAM8 were found to be pivotal for the regulation of granulocyte activation in humans [16] . While human CEACAM1 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic domain, one CEACAM1 ITIM is replaced by an immunoreceptor tyrosine-based switch motif (ITSM) in mice and rats. Human CEACAM3 and possibly CEACAM4, CEACAM19 and CEACAM20 harbor functional immunoreceptor tyrosine-based activation motifs (ITAM) in their cytoplasmic tails [17, 18] . In contrast, except for CEACAM19 and CEACAM20 no such ITAM-containing CEA family members exist in rodents. Remarkably, several pathogens namely, Neisseria sp., Haemophilus influenzae, and Moraxella catarrhalis in humans [19] [20] [21] and mouse hepatitis virus (MHV) [22] use CEACAMs to anchor themselves to or to invade host cells. In addition, it has been suggested that bacterial pathogens can down-regulate immune functions by binding to CEACAM1 on CD4+ T lymphocytes and signaling via the CEACAM1 ITIM motif [23] . It was recently proposed that the human granulocyte-specific CEACAM3 functions as a specifically adapted single-chain ITAM-dependent phagocytic receptor involved in the clearance of CEACAM-binding bacteria by human granulocytes [24] . This indicates that fixation of genes in the human genome coding for ITAM-bearing CEACAMs can be the result of a selective pressure mediated by CEACAM-binding pathogenic microbes. In contrast, one of the two allelic variants of murine CEACAM1 exhibits a significant lower virus receptor activity suggesting that, in mice, mutations in the CEACAM1 gene rather than the fixation of an activating virus receptor were acquired to evade pathogen attack. The availability of a high-quality draft genome sequence of the domestic dog (Canis familiaris) [25] provided the possibility to analyze the complete CEA gene family in a third mammalian order. Here we show that in the dog multiple ITAM-bearing CEACAM1-related CEACAMs evolved and that CEACAM28 and CEACAM1 have undergone concerted evolution by gene conversion. Based on sequence analyses and comparison of expression profiles we suggest that CEACAM28 and CEACAM1 are paired activating and inhibitory immune receptors involved in the regulation of T cell responses. The possibility that sporadic recurrence of selective pressure mediated by species-specific pathogens was the driving force for the evolution of multiple ITAM-bearing CEACAM1-related CEACAMs in the dog is discussed.