Evaluation of Four Humanized NOD-Derived Mouse Models for Dengue Virus-2 Infection

Abstract

Dengue is a significant public health problem with no specific viral treatment. One of the main challenges in studying dengue is the lack of adequate animal models recapitulating human immune responses. Most studies on humanized mice use NOD-scid IL2R gamma null (NSG) mice, which exhibit poor hematopoiesis for some cell populations. This study compares three humanized (hu) NOD-derived mouse models for dengue virus-2 (DENV-2) infection in the context of human cytokine expression. Three mouse strains (hu-NSG, hu-EXL, and hu-SGM3) received xenotransplants of human CD34+ fetal cord blood cells from a single donor, and one mouse strain received human peripheral blood mononuclear cells (hu-SGM3-PBMCs). All models exhibited infectious viruses in blood confirmed by plaque assay, but mice expressing human cytokines showed higher viremia compared to conventional NSG mice. The hu-SGM3-PBMCs model developed lethal infections, showing a significant increase in viremia and clinical signs. A detectable human cytokine response was observed in all the DENV-2-infected humanized mouse models. In conclusion, humanized NOD-derived mouse models expressing human cytokines offer a relevant platform for the study of dengue pathogenesis and antiviral therapies.

Keywords: CD34+; EXL; NCG; NSG; PBMCs; SGM3; dengue; humanized mice.

Figure 1

Comparison of DENV−2 disease and replication in hu-NSG, hu-EXL, and hu-SGM3 mouse models. (A) Kaplan–Meier survival curves depicting the outcomes for hu-NSG (n = 6), hu-EXL (n = 8), and hu-SGM3 (n = 7) mice infected with 10⁷ PFU/mL via intravenous route. Two controls for each mouse model were used. Mice were closely monitored over a 20-day period. (B) Necropsy findings illustrating a hu-EXL-infected mouse’s demise on the 17th day post-infection, revealing bleeding and necrosis of the small intestine with the presence of lymph nodes (LN). (C) Viral kinetics in the hu-NSG model after xenotransplantation with CD34+ HSC. hu-NSG (DENV−2 1 × 10⁷ PFU) are infected mice; hu-NSG (C636 supernatant) are control mice, and NSG (DENV−2 1 × 10⁷ PFU) are non-humanized mice inoculated with DENV. (D) Viral kinetics in the hu-EXL model expressing human cytokines and xenotransplanted with CD34+ HSC. hu-EXL (DENV−2 1 × 10⁷ PFU) are infected mice; hu-EXL (C636 supernatant) are control mice, and EXL (DENV−2 1 × 10⁷ PFU) are non-humanized mice inoculated with DENV. (E) Viral kinetics in the hu-SGM3 model expressing human cytokines and xenotransplanted with CD34+ HSC. hu-SGM3 (DENV−2 1 × 10⁷ PFU) are infected mice; hu-SGM3 (C636 supernatant) are control mice, and SGM3 (DENV−2 1 × 10⁷ PFU) are non-humanized mice inoculated with DENV. At the upper section of each diagram, the count of positive samples is indicated for the total serum samples analyzed on each respective day. No statistical difference was observed in the survival curves between models (log-rank (Mantel–Cox) test p > 0.05) or viremia between hu-EXL and hu-SGM3 (Kruskal–Wallis p > 0.05).

Figure 2

Identification of infectious virus in RT-qPCR-positive serum samples from hu-mouse models (CD43+) and cytokine profile in hu-mice models infected with DENV−2 at 7- and 20-days post-infection. (A) Relation between plaque formation units (plaque assay) and genome equivalent (RT-qPCR) copies per mL observed in supernatants derived from the indirect plaque assay (n = 3); DENV−2 propagated in C636 was used as a control; *hu-EXL sample did not develop plaques yet still yielded a positive result when subjected to RT-qPCR. (B) Representative plaque assay images of samples from each model; DENV−2 propagated in C636 was used as a positive control, and RPMI media was used as a negative control. Human cytokine profile detected in (C) hu-NSG (CD34⁺), (D) hu-EXL (CD34⁺), and (E) hu-SGM3 (CD34⁺). Each plot displays data from three individual mice within their respective groups; values from control mice inoculated with C636 supernatant control sera were subtracted. At the upper section of each diagram, the count of positive samples is indicated for the total serum samples analyzed on each respective day.

Figure 3

Assessment of DENV−2 disease in hu-SGM3 (PBMCs), mouse model. (A) Kaplan–Meier survival curves depicting the outcomes for hu-SGM3 PBMCs (n = 8) mice infected with 10⁷ PFU via intravenous route. Two controls for each mouse model were used. Mice were closely monitored over a 20-day period. (B) Clinical manifestations captured in hu-SGM3 PBMC mice infected with DENV-2 at 10⁷ PFU/mL via the intravenous route on day 18. Infected mice exhibit diarrhea and a hunchbacked posture, while the controls displayed hair loss due to Graft versus Host Disease (GvHD). (C) Representative necropsy findings from hu-SGM3 PBMC mice (five out of eight mice), uncovering internal bleeding and small intestine necrosis. Control (C636 Supernatant) in C images corresponds to the mouse displaying the GvHD phenotype. The survival curves between hu-SGM3 (PBMCs), hu-NSG (CD34+), hu-EXL (CD34+), and hu-SGM3 (CD34+) showed a statistically significant difference (p < 0.001) log-rank (Mantel–Cox) test.

Figure 4

Dynamics of DENV−2 infection in the hu-SGM3 PBMCs model. Blood samples were collected, and subsequent serum samples were used for viremia and human cytokine assessment. (A) Viral kinetics in the hu-SGM3 PBMCs model. hu-SGM3 (PBMCs) (DENV−2 1 × 10⁷ PFU) are infected mice, and hu-SGM3 (PBMCs) (C636 supernatant) are control mice. (B) Relation between plaque formation units (plaque assay) and genome equivalent (RT-qPCR) copies per mL observed in supernatants derived from the indirect plaque assay (n = 3); DENV−2 propagated in C636 was used as a control. (C) Representative plaque assay images of indirect plaque assay, DENV−2 propagated in C636 were used as a positive control, and RPMI media was used as a negative control. (D) human cytokine profile in hu-SGM3 PBMCs model infected with DENV−2 at 7- and 18-days post-infection. Values from hu-SGM3 PBMCs inoculated with C636 supernatant control sera were subtracted. At the upper section of each diagram, the count of positive samples is indicated for the total serum samples analyzed on each respective day. ** p < 0.001 (Dunn test) when compared with the hu-EXL (CD34+) and hu-SGM3 (CD34+).