Aged AG129 mice support the generation of highly virulent novel mouse-adapted DENV (1-4) viruses exhibiting neuropathogenesis and high lethality

Abstract

Dengue virus infection in humans ranges from asymptomatic infection to severe infection, with ∼2.5 % overall disease fatality rate. Evidence of neurological manifestations is seen in the severe form of the disease, which might be due to the direct invasion of the viruses into the CNS system but is poorly understood. In this study, we demonstrated that the aged AG129 mice are highly susceptible to dengue serotypes 1-4, and following the adaptation, this resulted in the generation of neurovirulent strains that showed enhanced replication, aggravated disease severity, increased neuropathogenesis, and high lethality in both adult and aged AG129 mice. The infected mice had endothelial dysfunction, elicited pro-inflammatory cytokine responses, and exhibited 100 % mortality. Further analysis revealed that aged-adapted DENV strains induced measurable alterations in TLR expression in the aged mice as compared to the adult mice. In addition, metabolomics analysis of the serum samples from the infected adult mice revealed dysregulation of 18 metabolites and upregulation of 6-keto-prostaglandin F1 alpha, phosphocreatine, and taurocholic acid. These metabolites may serve as key biomarkers to decipher and comprehend the severity of dengue-associated severe neuro-pathogenesis.

Keywords: Aged mice; Cytokines; Dengue; Metabolomics; Pathogenesis; Toll-like receptor.

Fig. 1

DENV-age adapted viruses are highly virulent and lethal in adult AG129 mice. 6-8 weeks mice (n = 6) were infected intravenously with the age-adapted strains of dengue virus 1–4 serotypes. (A) Weight change, survival, and behavioral clinical scoring of DENV-1-Ad, DENV-2-Ad, DENV-3-Ad, and DENV-4-Ad were monitored for up to 14 days on a daily basis. (B) On 3^rd dpi infected spleen, serum, and brain were harvested and the titer was determined in Vero E6 cells by immunofluorescence assay from the infected mice. (C) NS5 gene-specific viral load determination, the spleen samples were collected in Trizol and processed for the viral load quantification in the infected spleen and serum as described in the Methods section. Statistical significance was calculated using one-way ANOVA for comparing control, adapted, and non-adapted strains of the DENV-infected group. Dunnett's multiple comparison test was used with alpha = 0.05. Where p < 0.05 was considered significant. *p < 0.05; **p < 0.001, ***p < 0.0001 and ****p < 0.00001.

Fig. 2

Assessment of vascular leakage in major systemic organs and brain viral load. 6–8 weeks old AG129 (n = 3) mice were intravenously injected with 200 µL of 10 MLMLD₅₀ of DENV-1-Ad, DENV-2-Ad, DENV-3-Ad and DENV-4-Ad virus. On 4^th dpi when mice started to show clinical signs of infection, three mice were administered 200 µL of 1 % Evans blue dye and after 45 min mice were euthanized in a CO₂ chamber and all the organs were harvested after extensive perfusion with PBS as described in the methods section. (A) Graphs of absorbance per gram tissue weight in peripheral organs. (B) Graphs of absorbance per gram tissue weight in infected brain perfused with 2 % Evan's blue dye. (C) Picture of the harvested brain showing retention of dye in adapted strains is compared with non-adapted strains and normal control mice and (D) On 3^rd dpi brain was harvested from DENV- 1–4 infected mice and the virus titer was determined in Vero E6 cells by immunofluorescence assay. Statistical significance was calculated using one-way ANOVA for comparing control, adapted, and non-adapted strains of the DENV-infected group. Dunnett's multiple comparison test was used with alpha = 0.05. Where p < 0.05 was considered significant. *p < 0.05; **p < 0.001, ***p < 0.0001 and ****p < 0.00001.

Fig. 3

Hematological parameters and histopathological manifestation in infected adult mice. 6–8 weeks old AG129 (n = 6) mice were intravenously injected with 10 MLD₅₀ age-adapted DENV viruses (DENV-1–4). (A–D) Blood parameters were monitored before infecting AG129 mice and on 3^rd and 7^th dpi except DENV-3 due to mortality at 5^th dpi. Samples were analyzed using the SS 22 VET Hematology analyzer on respective days as per the manufacturers’ instructions. C* In DENV-3-Ad infection mice survived till day 5 only. (E-H) Histopathological changes in liver and spleen of control mice, non-adapted DENV viruses and age-adapted DENV viruses’ infected mice. Black arrows in the liver sections show alteration of hepatocytes architecture, vacuole formation, haemorrhage in micro and macro-vesicular steatosis, and infiltration of mononuclear cells, and white arrows in spleen sections show a widening of white pulp. disruption of white pulp and red pulp ratio and splenic architecture. Scale bar = 50 µm. Statistical significance was calculated using one-way ANOVA. Student's t-test was used for comparing adapted virus-infected mice with uninfected control mice with alpha = 0.05. Where p < 0.05 was considered significant. *p < 0.05; **p < 0.001, ***p < 0.0001 and ****p < 0.00001.

Fig. 4

Aged-mouse adapted virulent DENV viruses’ induced release of inflammatory cytokines. (A–D) Serum samples were collected on 4th, 6th, and 8th dpi from DENV-1-Ad, DENV-2-Ad, and DENV-4-Ad and only on 4^th dpi in the case of DENV-3-Ad infected mice and control mice. The five different cytokines; i.e., IL-2, IL-4, IL-5, IFN-γ, and TNF-α were examined through the Th1/Th2 Cytokine Kit (BD Biosciences). C* In DENV-3-Ad infection mice survived till day 5 only. The cytokines level was acquired using the FACS Canto-II flow cytometer (BD Biosciences) and the acquired FACS results were generated in tabular and graphical format using CBA Analysis Software (BD Biosciences), Statistical tests were performed with n = 5 mice using 1- or 2- way ANOVA, and Dunnett post hoc test, Where, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 were considered significant.

Fig. 5

Differential TLR expression in aged-mouse adapted virulent DENV viruses in adult and aged mice (A) Schematic representation of relative versus absolute quantification of infected AG129 mice by real-time PCR. (B) Survival graph of infected adult versus aged AG129 mice, Adult (6–8 weeks old) and aged AG129 (>12-month-old) mice were perfused at 3^rd dpi, for spleen collection. (C) NS5 gene-specific viral load in the infected spleen of adult versus aged AG129 mice (D) Relative gene expression of TLR-3/7 and 9 in normal control adult versus infected adult AG129 mice and normal control aged versus infected aged AG129. Statistical significance was calculated using one- and two-way ANOVA for n = 5 control, adult, and aged AG129 mice infected with DENV-1-Ad, DENV-2-Ad, and DENV-4-Ad virus. Where, ***p < 0.0001 and ****p < 0.00001 were considered as a significant.

Fig. 6

Serum metabolites profiling of the adult mice infected with DENV-Ad and NAd challenge viruses. 2D-score plots of Principal Component Analysis (PCA) between the serum metabolites of (A) All the five groups used in the prevalent study; wherein PC1 and PC2 explain 29.4 % and 13.1 %, respectively. (B) Heat map representation of top 100 differentially expressed metabolites amongst all the study groups. The features highlighted with deep orange represent unregulated metabolites while those in deep blue represent the downregulated metabolites in each trait. (C) Table showing a list of common metabolites between DENV-1-Ad and DENV-2-Ad infected samples. Red: Highlights upregulated metabolite, Blue: Highlights downregulated metabolites.