Characterization of a murine model of non-lethal, symptomatic dengue virus infection

Abstract

The mosquito-borne disease dengue is caused by four serologically- and genetically-related viruses, termed DENV-1 to DENV-4. Historical setbacks due to lack of human-like mouse models of dengue were partially remedied with characterization of lethal DENV-2 infection in immunocompromised AG129 mice (deficient in IFN-α/β/γ receptors). Recently, our group established lethal AG129 mouse infection models of DENV-1, DENV-3, and DENV-4 using human isolates. Here we compare a non-lethal, disseminated model of DENV-3 infection using strain D83-144 to that of the lethal outcome following infection by strain C0360/94. Both strains belong to DENV-3 genotype II and differ by only 13 amino acids. Intraperitoneal inoculation of AG129 mice with strain D83-144 led to clinical signs of dengue infection, such as cytokine induction, thrombocytopenia, and systemic infection. However, C0360/94 infection led to features of severe human dengue, including coagulopathy and lethal outcome, whereas D83-144 infection does not. This study is the first to investigate a low passage, non-mouse lethal strain in AG129 mice and demonstrates that D83-144 infection induces milder features of human dengue than those induced by lethal C0360/94 infection. The results suggest that the AG129 mouse model has applications to investigate factors associated with mild or severe disease.

Figure 1

Replication of DENV-3 strains isolated from human cases in Thailand. In vitro kinetics of DENV-3 strains at a multiplicity of infection of 0.1 in C6/36 (A) or Vero (B) cells show that the strains have similar replication. Symbols: mean, error bars: standard error of the mean (SEM), n = 3; f.f.u.: focus forming units; limit of detection (L.O.D) 10² f.f.u.

Figure 2

Comparison of DENV-3 disease in adult AG129 mice. (a) Kaplan-Meier survival curves of mice infected with 10^7.0 (n = 11) or 10^7.7 (n = 9) f.f.u. of C0360/94 or 10^7.0 (n = 2), 10^7.5 (n = 3), or 10^8.0 (n = 4) f.f.u. of D83-144 via the i.p. route. Mice were monitored for at least 4 weeks. (b) Percent weight lost during infection relative to animal weight prior to infection, each line represents the time course of a single animal. (c–g) Mice inoculated with 10^7.5 f.f.u. D83-144 or C0360/94 were sacrificed daily to determine viral loads during acute infection. (c) Blood was collected and serum samples were used for viremia measurement; results are from three independent experiments. Differences in daily viral titers between D83-144 and C0360/94 were determined by Mann-Whitney tests and asterisks denote significance; day 2 p = 0.0003, day 3 p = 0.0011, day 1 was not significant, p = 0.0545 (denoted by #). (d–g) Internal organ samples were homogenized, and virus was titrated; results are pooled from two separate experiments. (d) Liver viral titers were significantly different on day 1 (p = 0.0030) and day 2 (p = 0.0466). (e) Spleen viral titers were significant on day 1 (p = 0.0061) and day 2 (p = 0.0012). (f) Viral titers in the large intestine were significant on day 2 only (p = 0.0344) and trended significant on day 3, p = 0.0669 (denoted by #). Viral titers in the brain were low for both D83-144 and C0360/94, but were significantly different on day 2 (p = 0.0128). Serum titers are expressed as log₁₀ f.f.u./ml and organ titers as log₁₀ f.f.u./g of tissue. Symbols represent mean sample titers, error bars: SEM. L.O.D. as follow: serum: 10^2.0 f.f.u.; liver: 10^2.8 f.f.u.; spleen: 10^2.8 f.f.u.; large intestine: 10^3.0 f.f.u.; brain: 10^2.7 f.f.u.

Figure 3

Histopathology during D83-144 infection. (a–i) Representative slides from D83-144-infected mice at 2 and 3 days post-infection (p.i.) are shown at 10× and 40× magnification. Controls for comparison are from liver sections from 2 day p.i. mock-infected mice, shown as control (a,b). At 2 and 3 days p.i. (d,e,g and h, respectively) D83-144 infection leads to activation of hepatocytes shown as binucleation or nuclear pleomorphism. To detect active replication in the liver, sections were immunostained with rabbit-anti-DENV-NS3, as described in Methods. Positive (red) signal was observed for DENV NS3 in the D83-144 2 (f) and 3 (i) days p.i. slides, but not in the control antibody-stained slide (c); DAPI was used to stain nuclei (blue). Further, control (j) or 2 day p.i, (k) spleen samples were stained for NS3 (positive red stain on day 2). Splenomegaly in D83-144 was calculated as a function of spleen mass to total body mass (g) and compared with ANOVA and Dunnett’s post-test: day 1 p = 0.0029, day 2–4 p < 0.0001. Results are from two independent experiments; day 0 and 1, n = 4; day 2 and 3 n = 7; day 4 n = 2.

Figure 4

Chemistry profile of D83-144-infected mice. Vetscan was used to analyze Lithium Heparin anticoagulated blood that was harvested from mock- or virus-infected mice sacrificed on day 1 or 3 p.i.; mock: n = 3 both days, D83-144: n = 2 day 1 and n = 6 day 3. (a) Comprehensive diagnostic panel rotor analysis of samples. Individual values are represented by symbols and sample means depicted by lines. Asterisks denote significance of D83-144 blood samples compared to uninfected controls using t-tests. Results are pooled from two experiments. (b) Samples collected on day 3 after mock (n = 4), D83-144 (n = 3), or C0360/94 (n = 4) infection were analyzed by Mammalian liver profile rotor tests. Bars represent the mean, error bars are SEM, and the three groups were compared using ANOVA with Tukey’s post-test; significant p-values are depicted in the graphs.

Figure 5

Blood profile of D83-144-infected mice. (a) Levels of platelets, percent hematocrit, leukocytes, and lymphocytes were determined from EDTA-anticoagulated blood from control (n = 10) or D83-144-infected mice on day 1 (n = 5), day 2 (n = 7), or day 3 (n = 7). Results are pooled from two separate experiments; lines represent the mean daily value, and comparisons between control and infected samples were determined with ANOVA with post-test. (b) Effects of D83-144 or C0360/94 infection on coagulation were determined on day 3 p.i. using the Vetscan PT/aPTT test (prothrombin time/activated partial thromboplastin time) on sodium citrate-anticoagulated blood from mock- (n = 4), D83-144- (n = 3) or C0360/94-infected (n = 4) mice.

Figure 6

D83-144 infection with 10^7.5 f.f.u. leads to altered serum cytokine levels. Sera harvested from naïve (n = 10) or D83-144-infected mice (day 1 n = 7, day 2 n = 6, day 3 n = 7, day 4 n = 2) were analyzed using Bioplex. Bars represent the mean; error bars are the SEM. Daily changes in values of infected samples for 1-4 dpi were compared using Kruskal-Wallis ANOVA with post-test, and statistical significance is depicted with asterisks above the columns as follow: ****p < 0.0001, ***0.0001 < p < 0.001, **0.001 < p < 0.01, *0.01 < p < 0.05. For day 4 p.i. group, significance is denoted by a distinct symbol (∝) to highlight that analyses were from n = 2; some control values were not determined (n.d.) for some cytokines because the levels were undetectable.

Figure 7

Bayesian phylogenetic analysis of nucleotides 38-10606 of representative strains from DENV-3 genotypes I (purple clade), II (green clade) and III (orange clade), and DENV-2 16681 (outgroup). Posterior probabilities of major nodes are included. C0360/94 and D83-144 are in red and blue, respectively. Strains are shown with accession number, country, and year of isolation. When multiple strains were available, only one strain per country per year was included in the analysis. Country abbreviations: Anguilla (AI), Australia (AU), Bangladesh (BD), Brazil (BR), Cambodia (CA), China (CH), Colombia (CO), Cook Islands (CK), East Timor (TL), Ecuador (EC), French Polynesia (PF), Guyana (GY), India (IN), Indonesia (IO), Martinique (MA), Mexico (MX), Mozambique (MZ), Nicaragua (NI), Pakistan (PK), Paraguay (PA), Peru (PE), Philippines (PH), Samoa (WS), Saint Lucia (LC), Singapore (SG), Sri Lanka (LK), Taiwan (TW), Thailand (TH), Trinidad and Tobago (TT), United States of America-Puerto Rico (US), Venezuela (VE), Vietnam (VN).

Figure 8

Location of potential molecular determinants of DENV-3 based on solved structures in E protein, NS3 Helicase, and RNA dependent RNA polymerase. (a) The E protein ectodomain homodimer cartoon is shown with ED1 in red, ED2 in yellow, and ED3 in blue. The D83-144 amino acids are depicted as sticks in green: Ser 124, His 132, and Ile 172. (b) The DENV-4 NS3 Helicase protein cartoon is shown in purple with a nucleic acid (orange), a nonhydrolysable ATP analogue, AMPPNP, in blue, and Lys 398 (equivalent to DENV-3 D83-144 Lys 398) as a stick in green. (c) The DENV-3 NS5 RNA dependent RNA polymerase cartoon is shown in salmon with the C0360/94 amino acid Thr 339 and D83-144 amino acid Arg 389 in green sticks.