This study was approved by the Institution Review Board (IRB) of Chang Gung Memorial Hospital-Kaohsiung Medical Center, Taiwan. The dengue patients were recruited as described previously in the 2002–2003 DEN-2 outbreak in Taiwan ^[19]–^[23]. A larger retrospective cohort was designed and re-approved by an additional IRB review (Document No.: 97-2111B). To validate cell surface expression and immune functions of rs4804803 SNP, we obtained informed consent to collect blood leukocytes from normal volunteers with AA or AG genotypes of rs4804803.

DEN infection was confirmed by clinical dengue symptoms and signs along with detection of DEN-2 RNA by quantitative RT-PCR in blood, detection of IgM to DEN or at least a 4-fold increase in dengue-specific hemagglutination inhibition titers in convalescent serum compared with that in acute-phase serum ^[20], ^[21]. In those with DEN-2 infection, blood was drawn at least once a day subsequent to admission into the hospital to measure the platelet counts and hematocrit levels. A Chest X-ray and abdominal ultrasonography were performed routinely in individuals without evidence of hemoconcentration or hypoalbuminemia to refine the differential diagnosis of DHF vs. DF based on pleural effusion or ascites. A clinical diagnosis of DHF was assigned according to the DHF criteria of the World Health Organization (WHO); including a reduced platelet count (<100,000/mm³), petechiae, hemorrhagic manifestation, and plasma leakage showing hemoconcentration (peak hematocrit ≥20% above the mean for the population, or an increase in hematocrit of 20% or more), pleural effusion, ascites, or hypoalbuminemia ^[24]. Patients with DF were defined by detectable DEN-2 RNA by RT-PCR or DEN-specific IgM, but without evidence of DHF. Primary or secondary DEN infections were identified using previously established serologic criteria for IgM/IgG ELISAs ^[19].

Patients with other non-dengue febrile illnesses (OFI) were defined by febrile illness with no detectable DEN-specific IgM, no detectable DEN RNA, and no obvious or reported bacterial etiology for their illness during the same study period. Population controls were healthy, unrelated volunteers from the same community, with neither signs nor previous history of dengue infection, with a DEN IgG sero-positive rate of 1.37% (1/73).

Genomic DNA was isolated from heparin-anticoagulated blood samples using a standard phenol-chloroform extraction followed by 70% alcohol precipitation. Genotyping for the CD209 variant (−336 A/G; rs4804803) was carried out using Custom TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA). The primer sequences were [gene: 5′-GGACAGTGCTTCCAGGAACT-3′] (forward) and [gene: 5′-TGTGTTACACCCCCTCCACTAG-3′] (reverse). The TaqMan minor groove binder probe sequences were [gene: 5′-TACCTGCCTACCCTT G-3′], and [gene: 5′-CTGCCCACC CTTG-3′]. The probes were labeled with the TaqMan fluorescent dyes VIC and FAM, respectively. The PCR was conducted in total volume of 15 µL using the following amplification protocol: denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 20 s, followed by annealing and extension at 60°C for one minute. After the PCR, the genotype of each sample was determined by measuring the allele-specific fluorescence in the ABI Prism 7500 Sequence Detection System, using SDS 1.1 software for allele discrimination (both Applied Biosystems). To validate the genotyping by real-time PCR analysis, 100 PCR products were subject to restriction fragment length polymorphism (RFLP) analysis with MscI restriction enzyme (New England Biolabs, Beverly, MA, USA) and showed 100% identical result between these two genotyping systems.

Peripheral blood mononuclear cells were collected from peripheral blood of 20 healthy, DEN-specific IgM or IgG seronegative volunteers with AA or AG genotype. CD14⁺ monocytes were isolated by positive selection according to the manufacturer's specifications using CD14 microbeads and a magnetic cell separator (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched CD14⁺ cells (purity>95%) were cultured for 6 days in six-well plates in complete RPMI medium in the presence of 10 ng/mL rhGM-CSF and 5 ng/mL rhIL-4 at 37°C, and 5% CO_2. On day 3, half of the medium was replaced with fresh medium supplemented with rhGM-CSF and rhIL-4. Expression of markers was measured by flow cytometer using specific antibodies and their corresponding isotype controls.

Unless otherwise stated, monocyte-derived dendritic cells (MDDCs) were infected with DEN-2 at a multiplicity of infection (MOI) of 5 for 2 h at 37°C and 5% CO₂. Cells were washed twice to remove cell-free virus, and cultured in complete RPMI medium (without cytokines) at a density of 2×10⁵ cells/ml in 48-well plates. Cells and supernatants were removed and analyzed at 24, 48, and 72 h post-infection. For the neutralization experiments, cells were incubated in the medium alone or in the medium with the addition of anti-human CXCL10/IP-10 antibody (R&D Systems, Minneapolis, MN, USA) at 10 µg/mL for 30 min. Cells and supernatants were harvested and analyzed 24 h post-infection.

Total RNA extracted from MDDCs was subjected to quantitative RT-PCR to assess levels of mRNA corresponding to CD209 and ß2–microglobulin (B2MG) using the ABI PRISM 7500 instrument (Applied Biosystems). The forward primer, reverse primer sequence for detecting CD209 and B2MG were [gene: 5′-AACAGCTGAGAGGCCTTGGA-3′], [gene: 5′-GGGACCATGGCCAAGACA-3′], and [gene: 5′-AATTGAAAAAGTGGAGCATTCAGA-3′], [gene: 5′-GGCTGTGACAAAGTCACATGGTT-3′], respectively. The PCR cycling parameters were 40 cycles of PCR reactions at 94°C for 20 s, and 60°C for one minute. The results were detected in real-time and recorded on a plot showing fluorescence vs. time. RT-PCR products were also visualized on ethidium bromide-stained 1.5% agarose (Pierce Co., Rockford, IL, USA) gel with a 100- bp ladder (Pharmacia Biotech, Piscataway, NJ, USA) as a reference.

To measure the CD209 cell surface expression, MDDCs were stained with FITC-conjugated mAbs specific for DC-SIGN (R&D Systems, Minneapolis, MN, USA). An isotype-matched FITC-labeled control, mouse IgG2b (clone MOPC195, Immunotech, Beckman Coulter, Fullerton, CA, USA) was included in each experiment.

Total RNA extracted from MDDCs was subjected to assess DEN-2 RNA viral copies. Fluorescent RT-PCR was performed in an ABI 7500 quantitative PCR machine (Applied Biosystems) for 40 cycles using TaqMan technology as previously described ^[21].

Cytokine/chemokine production and viral replication were determined at 24, 48, and 72 h post-infection. Cell-free culture supernatants TNFα and macrophage chemoattractant protein 1 (MCP-1) concentrations were measured using ELISA kits from eBioscience Inc. (San Diego, CA, USA); IL-12p40 and IFN-inducible protein 10 (IP-10) concentrations were measured using ELISA kits from R&D Systems as per manufacturer's instructions.

Data are presented as mean ± SEM values. Alleles and genotypes distribution of rs4804803 are presented as numbers (percentages). Conformance of the allele frequencies to Hardy-Weinberg equilibrium proportions was tested to compare the observed and expected frequencies of heterozygotes and homozygotes. Differences among patients with DEN, DF, DHF, OFI, and population controls were determined using two-sided Chi-Square test or Fisher exact test. Odds ratio (OR) values were calculated with 95% confidence intervals (CI). The Student's t-test or Mann-Whitney U test was used for statistical comparisons between continuous variables. The Wilcoxon signed-rank test was used for statistical comparison of the neutralization experiments. All analyses were performed using SPSS 13.0 (SPSS Inc. Chicago, IL, USA).

During a large DEN-2 outbreak in southern Taiwan between June 2002 and January 2003, a hospital-based case-control study was used to identify the risk immune parameters ^[19]–^[23]. Employing the decoded DNA samples from that same cohort of the population that study has been extended to investigate the association of rs4804803 SNP with DF, DHF, viral replication, and immune response. Based on the previous case-control study design, we have included DNA samples from 135 DHF, 176 DF, and 143 OFI patients in this expanded study. The main characteristics of the study population are summarized in Table 1. There were no significant differences in sex or total leukocyte counts between patients with DF and those with DHF. However, age (41.7±1.6 years vs. 45.7±1.3 years, p<0.001), serum GOT levels (70.1±8.1 U/mL vs. 313.8±74.6 U/mL, p = 0.002) and GPT levels (67.1±11.3 U/mL vs. 142.7±21.9 U/mL, p = 0.003) were significantly higher in the DHF group (Table 1). A patient manifested with abdominal pain had ascites as evidenced by abdominal ultrasonography was classified as DF because the patient revealed no thrombocytopenia (<100,000/mm³), petechia or hemorrhagic manifestation during the admission period.

We investigated the association of rs4804803 SNP in the promoter region of CD209 with protection from dengue infection and the susceptibility of DHF. Genomic DNA obtained from DEN patients (n = 311), OFI patients (n = 143), and population controls (n = 120) was genotyped for rs4804803 SNP. We found that GG/AG genotypes in 16.0% of the DEN patients were significantly higher than OFI patients (5.6%, OR = 3.23, p = 0.002) and population controls (7.5%, OR = 2.36, p = 0.020; Table 2). Moreover, the GG/AG genotypes were significantly higher in DHF patients (23.0%) than OFI patients and population controls (OR = 5.03 and 3.68, p = 3×10⁻⁵ and 0.001), and also significantly higher than DF patients (10.8%, OR = 2.46; p = 0.004; Table 2).

Analysis of the allele distribution between DEN and OFI patients or population controls showed that the G allele frequency was higher in DEN patients (8.4%), compared with OFI patients (2.8%, OR = 3.17, p = 0.002) or population controls (3.8%, OR = 2.34, p = 0.018; Table 3). Moreover, the frequency of G allele of rs4804803 was significantly higher in DHF patients (12.2%) than OFI patients or population controls (OR = 4.84 and 3.57, p = 2×10⁻⁵ and 0.001), and higher than DF patients (5.4%, OR = 2.44, p = 0.002; Table 3). Few DHF patients (n = 6) had dengue shock syndrome in this cohort; one of them carrying AG genotype.

To investigate whether the rs4804803 SNP in the promoter region of CD209 associated with the primary and secondary DEN infection, we used serological methods to detect DEN antibodies for differentiation into primary and secondary dengue infection. Of the 293 DEN patients, 141 (48%) had secondary DEN infections, based on detectable DEN-2 virus RNA and DEN IgG. As shown in Table 1, secondary DEN-2 infection was more frequently found in patients with DHF than in those with DF (65% vs. 36%, p<0.001). We found the rs4804803 GG/AG genotypes were found in 12.5% of patients with primary DEN infection and 16.3% of patients with secondary DEN infection, which did not reach significantly different (OR = 1.36; p = 0.352). As shown in Table 4, there was no association between rs4804803 SNP and primary or secondary dengue infection in DF patients (OR = 0.64; p = 0.409), or in DHF patients (OR = 1.80; p = 0.251). In addition, there was no association between allele distribution and primary or secondary dengue infection in DF and DHF patients (data not shown).

Due to the low frequency of GG genotype in our population (2 cases, 0.6%), we could not recall the patients because the data file had been decoded for identification. We examined DC-SIGN (CD209) expression in both mRNA level of MDDCs and protein level on their cell surface from healthy subjects with AA or AG genotype by quantitative RT-PCR and flow cytometry, respectively. A significant increase in CD209 mRNA expression was detected in the MDDCs from individuals with AG genotype than those from individuals with AA genotype (p = 0.032, Fig 1B). Similarly, individuals with AG genotype had a significantly higher cell surface DC-SIGN expression (p = 0.029; Fig 1D). However, the surface DC-SIGN expression declined rapidly along with DEN-2 infection on MDDCs from both genotypes' subjects, which showed no difference at 24, 48, and 72 h post-infection (Fig. 2A).

To investigate whether the rs4804803 SNP was correlated to viral replication, we measured DEN-2 RNA copies in MDDCs with AA or AG genotype of rs4804803 at 24, 48, and 72 h post-infection. DEN-2 replication was significantly higher in MDDCs from individuals with AA genotype than those with AG genotype at 48 h post-infection (1.07±0.45×10⁶ copies/10⁵ cells vs. 3.90±0.67×10⁶ copies/10⁵ cells, p = 0.006) and 72 h post-infection (4.83±0.70×10⁵ copies/10⁵ cells vs. 2.32±0.68×10⁶ copies/10⁵ cells, p = 0.003; Fig. 2B). Viral replication, as measured at 72 h post-infection, increased more remarkably in MDDCs at MOI of 5 and 10 (p<0.001 and 0.002, respectively; Fig. 2C).

To investigate whether higher cell surface DC-SIGN expression was correlated with immune response, we investigated kinetic cytokine/chemokine production by MDDCs from individuals with AA or AG genotype of rs4804803. Results showed that MDDCs with AG genotype had significantly higher TNFα production than those with AA genotype at 24 and 48 hr post-infection (303.51±66.75 pg/mL vs. 143.97±68.80 pg/mL and 202.35±19.35 pg/mL vs. 73.00±9.55 pg/mL; p = 0.021 and 0.002, respectively; Fig 3A). IL-12p40 production significantly increased by MDDCs with AG genotype than those with AA genotype at 24, 48, and 72 h post-infection (p<0.001, 0.007 and 0.001, respectively; Fig 3B). We also measured the concentration of two chemokines, MCP-1 and IP-10, which had been implicated in the recruitment and stimulation of monocytes, macrophages, dendritic cells, NK cells, and T lymphocytes ^[25]. It was found that IP-10, but not MCP-1, production was significantly higher by MDDCs with AG genotype than those with AA genotype at 24, 48, and 72 h post-infection (620.60±175.56 pg/mL vs. 243.02±41.64 pg/mL, 889.92±91.46 pg/ml vs. 168.02±24.02 pg/mL, and 614.44±49.16 pg/mL vs. 322.32±69.62 pg/mL; p = 0.034, 0.009 and 0.010, respectively; Fig 3C). The MCP-1 levels peaked at 48 hr in subjects with both genotypes', but there was no significant difference between AA genotype and AG genotype (550.72±60.73 pg/mL vs. 463.92±66.80 pg/mL, p = 0.157; Fig. 3D).

IP-10, produced by non-infected bystander DCs in response to DEN infection, is a potent chemoattractant for activated T and NK cells ^[26], and the modulation of adaptive immune response ^[27]. IP-10 has also been known to inhibit the binding ability of DEN in immortalized cells ^[28]. In our MDDC model, cells from individuals with AG genotype exhibited an augmented innate immune reaction, showing higher IP-10 production, post-infection (Figure 3C). Based on these results, we hypothesized that DEN-infected MDDCs with AG genotype produced higher levels of IP-10, which might block viral entry or viral replication in MDDCs. We used an anti-IP-10 neutralizing mAb to block endogenous IP-10 production by MDDCs. With both genotypes, the viral replication 24 h post DEN infection increased significantly more in the presence of neutralizing antibody than in the absence of neutralizing antibody (p = 0.034 and 0.040, respectively; Fig. 4A). IP-10 production by MDDCs from individuals with AG genotype significantly decreased (795.3±368.1 pg/mL vs. 273.8±87.8 pg/mL; p = 0.037), but it did not decrease in MDDCs from individuals with AA genotype (329.8±114.2 pg/mL vs. 201.8±87.0 pg/mL; p = 0.091; Fig. 4B). These results suggest that IP-10 produced by MDDCs is involved in the viral replication of DEN infection.