Dengue virus in the heart.

<p>(a–c) Detection of DENV-3 antigens in general (a, b and c) and specifically the NS3 protein (e and f), by immunochemistry, in cardiac fibers (CF), endothelium (En) and monocytes (Mo). (d) Quantification of cells presenting dengue antigens. (g–i) Detection of virus RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (g and i, respectively). (h) One non-dengue case incubated with the probe. Arrows indicate positive staining in endothelium (En) and cardiac fibers (CF).</p

Dengue virus in the liver.

<p>(a–b) Detection of DENV-3 antigens in general, by immunochemistry, in hepatocytes (H), Kupffer cells (KC) and endothelium (En). (c) Quantification of hepatocytes and Kupffer cells presenting dengue antigens. (d) Detection of dengue NS3 protein by immunochemistry in hepatocytes (H), Kupffer cells (KC) and endothelium (En). (e–g) Detection of DENV-3 RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (e and g, respectively). (f) One non-dengue case incubated with the probe. Arrows indicate positive staining in hepatocytes (H).</p

Dengue virus in the spleen.

<p>Detection of DENV-3 antigens in general (a) and specifically the NS3 protein (c), by immunochemistry in macrophages (Mφ). (b) Quantification of cells presenting dengue antigens. (d–f) Detection of virus RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without or with the probe (d and f, respectively). (e) One non-dengue case incubated with the probe. Arrows indicate positive staining in macrophages. (g) Co-localization of the virus RNA negative strand (fluorescent blue) and CD68 (fluorescent red), for identification of macrophages. Cells presenting both staining showed yellow fluorescence.</p

Histopathological and ultrastructural analysis of the lung.

<p>(a) Lung of a non-dengue case stained with HE and presenting normal aspect of alveoli (A) and alveolar septa (AS). (b–g) Lung sections of dengue cases, stained with HE, showing pulmonary alterations, including septal thickening (St), edema (E), hemorrhage (He), presence of mononuclear infiltrate (Inf), hyaline membrane formation (HM) and hypertrophy of alveolar macrophages (AM) and type II pneumocytes (PcyII). (h) Semi-thin section of a non-dengue case showing alveoli (A), alveolar septa (AS), endothelial cells (EC) and type I (PcyI) and II pneumocytes (PcyII) with normal aspects. (i) Semi-thin section of one dengue case presenting numerous platelets (Pt) and megakaryocytes (MK) inside alveolar septa. (j) Ultrathin section of one non-dengue case exhibiting regular alveoli, alveolar septum, type I and II pneumocytes and endothelial cell. (j-l) Ultrathin sections of dengue cases exhibited type II pneumocytes located in alveolar space in contact with numerous platelets, the appearance of hyaline membrane and the presence of virus particles (VP) in endothelium. Quantitative analysis of hemorrhage (m) and edema (n) observed in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083386#pone-0083386-g001" target="_blank">figure 1</a>, as well as damage quantifications.</p

Histopathological/ultrastructural analysis and dengue detection in the kidney.

<p>(a) Kidney of a non-dengue case stained with HE and presenting normal aspect. (b and c) Kidney sections of dengue cases, stained with HE, showing injuries, including: hemorrhage (He), edema (E), sloughing of necrotic cells with loss of the basement membrane (black star), mainly in proximal convoluted tubule (PCT) but also detected in distal convoluted tubule (DCT), and areas of cellular regeneration (blue star) near renal glomerulus (RG). (d and e) Semi-thin sections of non-dengue cases showing Bowman’s capsule (BC) and podocytes (Pdc) around glomerular capillaries (GC), mensagial cells (MC) and endothelial cell (EC) with regular structures and preserved capillaries (Cap), epithelial cells (Ep) and distal and proximal convolutes tubules (DCT and PCT, respectively). (f and g) Dengue cases with the presence of thrombus (T) in capillaries of renal glomerulus and necrotic cells (NC) in the lumen of proximal convoluted tubules. (h) Ultrathin of a non-dengue case exhibiting conserved glomerular capillaries. (i) Ultrathin of one dengue case showing necrotic cell with picnotic nucleus (white star) and dilatation of rough endoplasmic reticulum (ER). Quantitative analysis of hemorrhage (j) and edema (k) observed in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083386#pone-0083386-g001" target="_blank">figure 1</a>, as well as damage quantifications. (l) Detection of DENV-3 antigens by immunochemistry in macrophages (M□) and monocytes (Mo) and (m) quantification of cells presenting these antigens.</p

Hospital and necropsy records from the four dengue fatal cases.

<p>ND: not determined.</p

Histopathological and ultrastructural analysis of the liver.

<p>(a) Liver of a non-dengue case stained with HE and presenting normal aspect. (b–e) Liver sections of dengue cases, stained with HE, showing hepatic injuries, including micro (Mi) and macrovesicular (Ma) steatosis, necrosis (N), edema (E) and hemorrhage (He) near central vein (CV). (f) Semi-thin section of a non-dengue case presenting hepatocytes and sinusoidal capillaries with normal structures and (g) one dengue case presenting micro (Mi) and macrosteatosis (Ma), nuclear degeneration (black star) and numerous macrophage cells (Mø). (h) Ultrathin section of a non-dengue case exhibiting normal hepatocytes (H) and regular sinusoidal capillaries (SC) with the presence of monocytes (Mo) and Kupffer cells (KC) and (i and j) dengue cases showing large lipid droplets (LD) in the cytoplasm of hepatocytes, swollen mitochondria (red stars) and presence of platelet (Pt) inside sinusoidal capillaries (SC) with loss of endothelium. Semi-thin and ultrathin sections of liver were stained with methylene blue/azure II solution and uranyl acetate/lead citrate, respectively. Quantitative studies of histological damages were made individually in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis were performed comparing the mean values of each group (dengue patients vs non-dengue patients). Damages were quantified by the percentage of affected area for (k) hemorrhage and (l) edema or (m) by steatosis degree using a scale ranging from 0 to 4. (n–o) Steatosis was also evaluated in each hepatic acini area (periportal, midzonal and central vein) by plotting different damage degrees (ten fields for each case). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05) and (***) (P<0.0001).</p

Dengue virus in the lung.

<p>(a, b, d and e) Detection of DENV-3 antigens in general (a and b) and specifically the NS3 protein (d and e), by immunochemistry, in macrophages (Mφ), type II pneumocytes (PcyII) and endothelium (En). (c) Quantification of cells presenting dengue antigens. (f–i) Detection of DENV-3 RNA negative strand by <i>in situ</i> hybridization. Dengue cases were treated without (g) or with the probe (h and i). (f) One non-dengue case incubated with the probe. (j) Co-localization of the virus RNA negative strand (fluorescent blue), by <i>in situ</i> hybridization, and CD31 (fluorescent red) for identification of endothelial cells and (k) cytokeratin AE1/AE3 (fluorescent green) for detection of pneumocytes, by immunohistochemistry. Cells presenting both staining (blue and red or blue and green) showed in yellow fluorescence.</p

Histopathological and ultrastructural analysis of the heart.

<p>(a) Heart of a non-dengue case stained with H.E. and presenting normal aspect. (b and c) Heart sections of dengue cases, stained with HE, showing cardiac injuries, including hemorrhage (He), edema (E), presence of mononuclear infiltrate (Inf) and degeneration of muscle fibers (black star). (d and f) Semi-thin and ultrathin sections of a non-dengue case presenting cardiac fibers (CF) with normal nucleus (N), mitochondria (M), capillaries (Cap) and intercalated discs (ID). (e) Semi-thin section of one dengue case presenting degeneration of cardiac fibers (black star) characterized by absence of nucleus and a diffuse interstitial edema (E) and (g and h) ultrathin sections showing nuclear (white stars) and mitochondria alterations (M) in cardiomyocytes and interstitial edema. Quantitative analysis of hemorrhage (i) and edema (j) observed in dengue (cases 1–4) and non-dengue patients (cont. 1–4), and statistical analysis performed comparing the mean values of each group (dengue patients vs non-dengue patients). Asterisks indicate differences that are statistically significant between control and dengue groups, (*) (P<0.05). Semi-thin and ultrathin sections were stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083386#pone-0083386-g001" target="_blank">figure 1</a>, as well as damage quantifications. (Pt) platelets; (EC) endothelial cells.</p

Percentage of survival of Balb/c mice immunized with pE1D2 and pE2D2 and challenged with DENV2.

<p>Mice were i.m. immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized and pcTPA-injected mice followed the same virus infection procedure. Mice were daily monitored and deaths were recorded. Differences between pE1D2- and pE2D2-vaccinated animals were statistical significant (p = 0.0027), as well as between these groups and control animals (p<0.0001). Data represent compilation of two independent experiments, with groups of 10 animals in each test (n = 20).</p