A lethal model of disseminated dengue virus type 1 infection in AG129 mice

Abstract

The mosquito-borne disease dengue is caused by four serologically and genetically related flaviviruses termed DENV-1 to DENV-4. Dengue is a global public health concern, with both the geographical range and burden of disease increasing rapidly. Clinically, dengue ranges from a relatively mild self-limiting illness to a severe life-threatening and sometimes fatal disease. Infection with one DENV serotype produces life-long homotypic immunity, but incomplete and short-term heterotypic protection. The development of small-animal models that recapitulate the characteristics of the disseminated disease seen clinically has been difficult, slowing the development of vaccines and therapeutics. The AG129 mouse (deficient in interferon alpha/beta and gamma receptor signalling) has proven to be valuable for this purpose, with the development of models of disseminated DENV-2,-3 and -4 disease. Recently, a DENV-1 AG129 model was described, but it requires antibody-dependent enhancement (ADE) to produce lethality. Here we describe a new AG129 model utilizing a non-mouse-adapted DENV-1 strain, West Pacific 74, that does not require ADE to induce lethal disease. Following high-titre intraperitoneal challenge, animals experience a virus infection with dissemination to multiple visceral tissues, including the liver, spleen and intestine. The animals also become thrombocytopenic, but vascular leakage is less prominent than in AG129 models with other DENV serotypes. Taken together, our studies demonstrate that this model is an important addition to dengue research, particularly for understanding the pathological basis of the disease between DENV serotypes and allowing the full spectrum of activity to test comparisons for putative vaccines and antivirals.

Keywords: AG129 mouse; Flavivirus; dengue virus; mouse model; pathogenicity.

Fig. 1.

DENV-1 WP 74 produces lethal infection in AG129 mice. (a) Survival curves of 6–8-week-old mice following intraperitoneal inoculation with 7.4 log₁₀ p.f.u. (n=19) or 6.4 log₁₀ p.f.u. (n=13) DENV-1 WP 74. Mice were monitored daily for 30 days. (b) Morbidity among these animals as measured by weight loss. Values are percentage change in body weight (mean±sd) relative to weight at challenge. (c) Survival curve for 18-week-old mice inoculated with 7.4 log₁₀ p.f.u. DENV-1 WP 74 (n=11). (d) Morbidity measured by weight loss in the 18-week-old animals.

Fig. 2.

DENV-1 WP 74 inoculation results in sustained viral RNAemia and virus dissemination to multiple organs. AG129 mice (6–8 weeks old) were inoculated with 7.4 log₁₀ p.f.u. DENV-1 WP 74. On days 1, 2 and 3 p.i., groups of five animals were sacrificed and serum and organs harvested. Organs were weighed and homogenized and virus load was determined by QRT-PCR. Results are shown for (a) serum, (b) liver, (c) spleen, (d) large intestine and (e) brain. Each symbol represents one animal. Serum titres are expressed as log₁₀ genome copies ml⁻¹ and organ titres are expressed as log₁₀ genome copies g⁻¹ of tissue. The dashed horizontal line represents the limit of detection of the assay. Symbols represent individual sample titres; short solid horizontal lines represent the mean daily titre.

Fig. 3.

DENV-1 WP 74 infection alters blood chemistry profiles. Six-week-old AG129 mice were inoculated with 7.4 log₁₀ p.f.u. DENV-1 WP 74 and blood was collected on days 1, 2 and 3 p.i. Samples were analysed using the Vetscan comprehensive diagnostic profile and the results were compared to those from media-inoculated control animals bled over the same period. Bars represent the mean (±sd) daily value. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (ANOVA with Dunnett's post test).

Fig. 4.

DENV-1 WP 74 infection impacts on multiple CBC parameters. Six-to-eight-week-old AG129 mice were inoculated with 7.4 log₁₀ p.f.u. DENV-1 WP 74 or served as uninfected controls (n=8). Blood was collected on days 2 (n=8) and 3 (n=7) p.i. Bars represent the mean (±sd) daily value. *P<0.05, **P<0.01 and ***P<0.001 (ANOVA with Dunnett's post test) compared to controls.

Fig. 5.

Histological changes resulting from DENV-1 WP 74 infection; H&E section micrograph images from AG129 mice mock-infected (a, c, e, g) or infected with 7.4 log₁₀ p.f.u. DENV-1 WP 74 (b, d, f, h) show the major changes seen in infected animals. (a, b) Ultrastructural changes in the virus infected spleen, with breakdown of normal architecture in the DENV-1-infected section. (c, d) Higher magnification images showing activated cells throughout the infected section. (e, f) Normal liver architecture in the control sections, but inflammation and focal necrosis in the DENV-1-infected section. (g, h) Areas of cellular expansion outside the Peyer’s patches in the large intestine of DENV-1-infected animals. Magnification: ×4 (a, b, e, f, g, h) or ×10 (c, d)

Fig. 6.

DENV-1 WP 74 infection results in alterations in the levels of cytokines and chemokines in serum. Serum was collected from 6 to 8-week-old AG129 mice (n=5/day) infected with DENV-1 WP 74 or age-matched media-treated controls (n=6). The levels of cytokines (a) and chemokines (b) were quantified from a panel of 23 cytokines using a multiplex bead assay (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocols. The cytokine abbreviations are as follows: IL, interleukin; KC, keratinocyte chemoattractant; G-CSF, granulocyte colony-stimulating factor; IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; MCP-1 (CCL2), monocyte chemoattractant protein-1 (CCL3 or 4); MIP, macrophage inflammatory protein; RANTES (CCL5), regulated on activation, normal T cell expressed and secreted; GM-CSF, granulocyte/macrophage colony-stimulating factor. *P<0.01, **P<0.001 (Student's t-test) compared to controls.

Fig. 6.

DENV-1 WP 74 infection results in alterations in the levels of cytokines and chemokines in serum. Serum was collected from 6 to 8-week-old AG129 mice (n=5/day) infected with DENV-1 WP 74 or age-matched media-treated controls (n=6). The levels of cytokines (a) and chemokines (b) were quantified from a panel of 23 cytokines using a multiplex bead assay (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocols. The cytokine abbreviations are as follows: IL, interleukin; KC, keratinocyte chemoattractant; G-CSF, granulocyte colony-stimulating factor; IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; MCP-1 (CCL2), monocyte chemoattractant protein-1 (CCL3 or 4); MIP, macrophage inflammatory protein; RANTES (CCL5), regulated on activation, normal T cell expressed and secreted; GM-CSF, granulocyte/macrophage colony-stimulating factor. *P<0.01, **P<0.001 (Student's t-test) compared to controls.