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

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

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

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

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

Laboratory findings and cardiac function measurements on the first study day in subjects with dengue.

<p>Values represent mean (SE), or number of cases.</p><p>^a two DHF cases did not have ultrasonography performed on the day of enrollment and were not included in this analysis. Differences between groups were analyzed by ANOVA with post hoc test or by Man Whitney’s test.</p><p>^<i>b</i>, ^<i>c</i> different from DHF without leakage at <i>P</i> < .005, and .05 respectively).</p><p>Laboratory findings and cardiac function measurements on the first study day in subjects with dengue.</p

Univariate correlations between indicators of disease severity and viral RNA levels in plasma and cell fractions collected on fever day-2.

<p>Correlations between viral RNA levels in plasma and cell fractions on fever day −2 and indicators of disease severity were analyzed by Spearman's Rho test. Multivariate non-parametric test of independence revealed that plasma viral RNA levels and viral RNA levels in CD14+ cell fraction independently correlated with pleural effusion index (P = .001).</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