Duration of DENV illness (days) by JEV NAb status.

*<p>Duration of illness was calculated as the number of days elapsed from first day of febrile illness to the last day that a child reported any fever, muscle or joint pain, headache, nauseas, vomiting, diarrhea, or any signs of bleeding or hemorrhage.</p>**<p>P-values were calculated using 2-way analysis of variance testing (ANOVA), with α = 0.05 as the level of significance.</p

Unadjusted associations of DENV illness severity and JEV antibody status.

*<p>Non-hospitalized infections incorporate both non-hospitalized DF and asymptomatic seroconversions.</p>**<p>Non-DHF infections incorporate both non-hospitalized and hospitalized DF and asymptomatic seroconversions.</p>†<p>P-values were calculated using the Mantel-Haenzel chi-square statistic.</p

Apoptosis related proteins in the thalamus of rhesus macaques after intranasal inoculation with JEV ((No. 2 (A, D, E), No. 9 (B), No. 11 (C)).

<p>(A) Some leukocytes in the perivascular infiltrates (left, arrowheads) and scattered unaltered appearing neurons (right; arrows) express cleaved caspase-3, an executor caspase. (B) The initiator caspase-8 is expressed by unaltered neurons (arrows) and some cells in the perivascular infiltrates (arrowheads). V: vessel. (C) Caspase-9, another initiator caspase, is expressed by microglial cells (arrowheads) and astrocytes (arrows). (D) Bax, a pro-apoptotic protein, is expressed by unaltered neurons (arrows) and microglial cells (arrowheads). (E) Bcl-2, an anti-apoptotic protein, is expressed by cells in the perivascular infiltrates. Indirect peroxidise method, DAB, Papanicolaou's hematoxylin counterstain. Scale bars = 50 µm.</p

Factors associated with JEV seropositivity among those experiencing dengue (DENV) infection.

*<p>Refers to first-detected DENV infections in the cohort that occurred during the active surveillance period each year (June 1 – November 1) and had neutralizing antibody data available.</p>**<p>Statistical tests considered the association between variables of interest and the presence or absence of JEV NAbs in the pre-infection sample. p-values were obtained using the Pearson chi-square test for categorical variables, with α = 0.05 as the level of significance. ‘Missing’ categories were not included in statistical comparisons.</p

DENV RNA levels in plasma and PBMC subpopulations in primary and secondary infections.

<p>Plasma and PBMC cell subpopulations of 11 primary and 15 secondary DENV infections, all of whom had DF, were analyzed. * indicates statistically significant differences in DENV RNA levels between primary and secondary infection (P<.05). +indicates differences between DENV RNA levels in CD20+ cells compared to CD14+ and CD2+ cells (P<.05).</p

Dengue virus in plasma and peripheral blood mononuclear cells.

<p>Comparisons of dengue viral (DENV) RNA levels in plasma and PBMC subpopulations in DF and DHF cases two days prior to defervescence (fever day −2) (A). * indicates statistically significant differences in DENV RNA levels between DF and DHF (P<.05). +indicates differences between DENV RNA levels in CD20+ cells compared to CD14+ and CD2+ cells (P<.05). (B, C, D, E) Levels of DENV RNA levels in plasma (B), CD14+ (C), CD2+ (D) and CD20+ (E) cells on fever day −2 and fever day −1. Each line represents individual cases: solid lines: DHF, dashed lines: DF.</p

Positive and negative strand DENV RNA levels in unfractionated PBMC.

<p>Quantitative PCR for positive and negative strand viral RNA was performed with RNA extracted from unfractionated PBMC collected on fever day-2 from subjects infected with DENV1 (A), DENV2 (B), DENV3 (C), or DENV4 (D). The range of the positive strand DENV RNA was 0 to 6069 copies/million cells. Negative strand DENV RNA was detected in 6 of 18 samples with detectable positive strand RNA and the levels ranged from 5 to 35 copies/million cells.</p

Cohort characteristics associated with symptomatic DENV illness.

*<p>Refers to first-detected DENV infections in the cohort that occurred during the active surveillance period each year (June 1–November 1) and had neutralizing antibody data available</p>**<p>Statistical tests considered the association between variables of interest and the occurrence of symptomatic versus asymptomatic infection. p-values were obtained using the Pearson chi-square test for categorical variables, with α = 0.05 as the level of significance. ‘Missing’ categories were not included in statistical comparisons.</p

Clinical severity of dengue infections by strata of preexisting DENV and JEV immunity.

<p>The proportions of dengue (DENV) infections resulting in symptomatic illness (1a), hospitalized illness (1b), and dengue hemorrhagic fever (1c) are shown. Data are stratified by preexisting DENV immunity (naïve [DENV -], monotypic [DENV 1+], multitypic older and younger than 10 years of age [DENV >1+]) and preexisting Japanese encephalitis virus (JEV) neutralizing antibodies (NAbs) (positive [+] or negative [-]). The JEV NAb positive groups are indicated by hash lines for each stratum of DENV immunity. Odds ratios (ORs) estimate the odds of being experiencing the disease severity of interest (dengue hemorrhagic fever [DHF], hospitalized illness [Hosp], or symptomatic illness [Sx]) in the presence of JEV NAbs over the odds of experiencing the disease severity of interest in the absence of JEV NAbs. Values in parentheses indicate the 95% confidence intervals for the ORs. Error bars indicate the 95% confidence intervals for proportions.</p

Inflammatory response in the thalamus of rhesus macaques after intranasal inoculation with JEV ((No. 2 (A, B, E) and No. 9 (C, D, F)).

<p>(A) CD3+ T cells dominate the perivascular infiltrates and are present in smaller numbers in the adjacent parenchyma (arrows). VL: vessel lumen. (B) CD20+ B cells represent a minority in the perivascular infiltrates. (C) Staining for CD68 identifies moderate numbers of macrophage/microglial cells within and surrounding the perivascular infiltrates (arrows) and highlights the large number of disseminated activated microglial cells in the adjacent parenchyma. (D) Macrophages in the perivascular infiltrates and the adjacent parenchyma (arrow) also express the myeloid/histiocyte antigen which indicates that they have recently been recruited from the blood. VL: vessel lumen. (E) Activated microglial cells also express major histocompatibility complex (MHC) class II antigen (arrowheads). MHC II is also expressed by vascular endothelial cells (arrows), confirming their activation. (F) Microglial nodule with central degenerate neuron (arrow), surrounded by CD68-positive microglial cells. Indirect peroxidise method, DAB, Papanicolaou's hematoxylin counterstain. Scale bars: A–E = 50 µm; F = 20 µm.</p