Weight loss curves of individual mice and splenocyte counts following i.n. VACV infection of C57/BL6 mice.

<p>(A) Groups of female C57/BL6 mice (n = 5–8) were infected with 10⁵, 10⁶ and 10⁷ PFU of vGK5 by the i.n. route. The percentage of weight relative to the initial body weight (100%) was plotted and the data are presented as percent change in body weight following infection. † depicts days that individual mice were last alive. (B) Average spleen counts±standard deviation of mice were assessed by trypan blue exclusion at days 3, 5 and 7 post infection with 10⁶ VACV-WR and vGK5 by the i.n. route. Data shown are representative of 2 experiments performed and demonstrate that high dose VACV-WR i.n. infections result in significantly (p<0.05) lower lymphocytes in the spleens during acute infection.</p

Immune responses following infection by the tail scarification and i.p. routes.

<p>Mice were infected with 1×10⁶ PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R_20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 10⁶ PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log₁₀ PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's <i>t</i> test.</p

Viral titers following i.n. infections.

<p>(A) Spleens and (B) lungs (n = 4–8 per group) were harvested on day 7 from mice infected with VACV-WR and vGK5 by the i.n. route. VACV titers were determined and expressed as log₁₀ PFU per gram of lung and spleen tissue. – represents median values of titers in respective organs. N.S. = not significant. Each symbol represents the titer obtained in target organs of individual mice; median values are denoted by horizontal lines. P values were determined by Student's <i>t</i> test.</p

Cytokine responses and cytolytic activity in target organs.

<p>(A) IFN-γ responses of splenocytes from intranasally infected mice were measured in response to 3 VACV-specific CD8 T cell peptides (1 µg/ml) in an Elispot assay. Assays were performed using triplicate wells for each condition and individual mice/group. * = no responses detected. Seven days post infection, (B) splenocytes and (C) lung lymphocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different effector to target (E/T) ratios. Data shown are 1of 2 experiments performed. (D) PRNT50 antibody titers were measured in the sera of mice immunized 5 months prior with varying doses of vGK5 or 10^3.5 PFU of VACV-WR (n = 5/group).</p

Mice immunized with vGK5 are protected from a lethal challenge with VACV-WR.

<p>Mice were infected with 10^4.5, 10⁶ PFU of vGK5 (n = 3/group) by the intranasal route. 1 month later immunized mice and age matched naïve controls were challenged with a lethal dose of VACV-WR (10⁶ PFU) by the i.n. route. (A) Weights of mice were monitored over 5 days. Viral titers were measured in the (B) lungs and (C) ovaries and expressed as viral titers/gm lung tissue or ovaries. Each symbol represents the titer obtained in target organs of individual mice.</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

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

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

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