Little is understood about the events that lead to DF or DHF after an infected mosquito (mainly Aedes aegypti) bites and injects virus into the human epidermis. The main targets for viral infection and replication seem to be dendritic cells (DCs) and macrophages, mainly Langerhans and monocyte-derived DCs (7, 13). A full repertoire of DCs develops in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice transplanted with human cord blood hematopoietic progenitor (CD34⁺) cells (4, 8). The NOD/SCID strain of mice lacks T and B cells and has defects in natural killer cell function and antigen-presenting cell development and function and genetically lacks C5, resulting in a deficiency in hemolytic complement; it therefore provides an excellent environment for reconstitution with human hematopoetic cells and tissues.

All animal procedures were reviewed and approved by our Institutional Animal Care and Use Committees.

CD34⁺ cells were isolated from human cord blood with immunomagnetic beads and transplanted into preconditioned mice (325 rads) as we have previously described (4, 8). Mice were analyzed for reconstitution with human cells (CD45⁺) and for the presence of human DCs (defined as CD11c⁺ or CD123⁺ lineage negative, HLA-DR⁺) in peripheral blood by flow cytometry using human-specific antibodies, as we have previously described (4, 8). We obtained high levels of reconstitution and DC development in multiple organs in these mice: human CD45⁺ cells in whole blood before inoculation (8+ weeks posttransplant) ranged from 0.9 to 43% (n = 18; median, 14%). A total of 18 reconstituted NOD/SCID mice were inoculated subcutaneously with approximately 4.7 log₁₀ PFU (7.7 log₁₀ genome equivalents) of dengue serotype 2 strain K0049 (Southeast Asia genotype; C6/36 cell passage 3); this virus was selected because it grows to high levels in human DCs in vitro (3). Six reconstituted and 15 nonreconstituted NOD/SCID mice (same genetic background but not receiving human CD34⁺ cells) served as negative controls (inoculated with saline, C6/36 cell culture supernatant, or virus). The most dramatic signs of dengue infection in humanized mice were erythema and thrombocytopenia (Fig. ​(Fig.1a),1a), as occurs in humans. We measured erythema using a DermaSpectrometer (Cortex Technology, Denmark) while mice were anesthesized; when compared to nonreconstituted, infected mice, erythema mean values were significantly different on days 1 through 8 (all pairwise t tests, P < 0.05; reconstituted mouse range, −1.36 to 8.44; nonreconstituted mouse range, −5.35 to −1.51). A marked decrease in platelets was measurable on day 8 postinoculation and was statistically highly significant (Mann-Whitney test, P < 0.001) when comparing six infected, reconstituted mice (range, 320,000 to 594,000/mm³) to three nonreconstituted, infected and six reconstituted, noninfected mice (normal mouse range, 600,000 to 1,200,000/mm³; range for our negative controls, 610,000 to 1,251,000/mm³). Rash was visible on days 2 to 4 in the majority (eight of nine) of infected, reconstituted mice and continued through day 14 in some mice (Fig. ​(Fig.1c).1c). Viremias were measured in sera (25- to 50-μl retro-orbital bleed on alternate days) by quantitative, real-time reverse transcription-PCR (RT-PCR) (Fig. ​(Fig.1b)1b) (11); virus levels peaked on days 2 to 6 postinoculation (range, 4.2 to 5.4 log₁₀ genome equivalents/ml). A rise in body temperature followed the viremia, and temperature returned to normal levels on day 10. For reconstituted mice, changes in temperature were statistically significant by analysis of variance (P < 0.02); for nonreconstituted mice, changes in temperature were not significant by analysis of variance (P 0.05). Nonreconstituted, infected NOD/SCID mice showed consistently lower viremias (undetectable by day 10; range, 3.6 to 4.1 log₁₀ genome equivalents/ml) and, as noted above, showed no rash, rise in temperature, or decrease in platelets and therefore served as negative controls for statistical analyses throughout. Body weight decreased dramatically in some reconstituted, infected animals (20% loss in four mice); there were no gastrointestinal abnormalities on necropsy, and weight loss seemed to be due to lethargy or lack of eating at the time of viremia or fever. Several organs were tested for viral positive- and negative-strand RNA template (surrogate for replicating virus) by quantitative, real-time RT-PCR (3) at different times postinfection in reconstituted and nonreconstituted mice. Only the reconstituted, infected mice had dengue virus RNA in various tissues on day 8: spleen (six of six mice), liver (three of six mice), and skin (one of six mice) had positive-strand RNA, while negative-strand RNA was detectable in spleen (two of six mice) and skin (one of six mice) (Fig. ​(Fig.2).2). These data are consistent with viral replication in this model.