Humanized mice show clinical signs of dengue fever according to infecting virus genotype

Abstract

We demonstrated that the infection of humanized NOD-scid IL2r gamma(null) mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34(+)). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45(+) cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10(6) PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.

FIG. 1.

Comparison of viremia levels in hu-NOD-scid IL2rγ ^null mice infected with four DEN-2 genotypes. (A) Mice were infected subcutaneously with 9 log₁₀ genome equivalents, and viral genome copies were determined in sera every 2 days by quantitative real-time RT-PCR. Shading highlights the comparison of viremias in mice infected with SE Asian genotype viruses (dark gray) against mice infected with American (B), Indian (C), and West African (D) (light gray) genotype viruses. Error bars indicate standard errors of the means, and an asterisk indicates a statistically significant difference (P = < 0.05) between the shaded groups. Results for the transplant-receiving mouse control group, inoculated with cell culture medium only, were always negative; one of six non-transplant-receiving mice inoculated with SE Asian strain K0049 had detectable virus (340 RNA copies/ml) at day 2 p.i.

FIG. 2.

Erythema indices in hu-NOD-scid IL2rγ ^null mice infected with four DEN-2 genotype viruses. (A) Erythema index was determined for infected and control mice (inoculated with medium) every other day starting at day 0 p.i., on the same shaved area of skin. (B) Erythema in groups infected with SE Asian (dark gray) and American (light gray) genotype viruses were compared. The erythema indices of mice infected with West African genotype (C) and Indian genotype (D) (dark gray) were compared with those of mice infected with the remaining three genotypes (light gray). Error bars indicate standard errors of the mean, and an asterisk indicates a statistically significant difference (P = < 0.05).

FIG. 3.

Temperature variation in hu-NOD-scid IL2rγ ^null mice infected with four DEN-2 genotype viruses. (A) Temperature was measured on infected and control mice (inoculated with media) every other day starting at day 0 p.i., using a rectal probe. (B) Comparison of temperatures of control group (dark gray area) against SE Asian, American, Indian, and West African (light gray area) genotype viruses. Error bars indicate standard errors of the mean, and an asterisk indicates a statistically significant difference (P = < 0.05).

FIG. 4.

Thrombocytopenia in hu-NOD-scid IL2rγ ^null mice infected with four DEN-2 genotype viruses. (A) Platelets were counted every 2 days in peripheral blood of mice infected with the SE Asian genotype, strain K0049; error bars indicate standard errors of the mean. (B) Comparison of platelet levels in mice infected with SE Asian, American, Indian, and West African genotype viruses at day 10 p.i. Error bars indicate standard errors of the mean, and an asterisk indicates a statistically significant difference (P = < 0.05).