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

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

JEV target cells in the thalamus of rhesus macaques after intranasal inoculation with JEV ((No. 7 (A, B), No. 2 (C–G)).

<p>(A) JEV antigen is seen in the majority of neurons (left: arrows). Right: Infected unaltered neurons express viral antigen in both cell body and cell processes. (B) JEV-infected neurons that are surrounded by microglial cells in satellitosis appear shrunken (arrows). (C) Microglial cells in particular in microglial nodules can be JEV-infected (top; arrow) and are identified based on their CD68 expression (bottom; arrow), as demonstrated in a consecutive section. (D) Dual staining for JEV antigen (FITC) and GFAP (Texas red) indicates that JEV does not infect astrocytes. (E) While endothelial cells (arrowheads) were not found to be JEV infected, perivascular macrophages in one animal were found to express JEV antigen (Texas Red); these cells were also undergoing apoptosis, since they were TUNEL-positive (FITC) (arrows). VL: vessel lumen. (F) Dual staining for JEV antigen (Vector Blue) and TUNEL (DAB) shows both the degenerating neurons and surrounding microglial cells in satellitosis undergo apoptosis (arrows). JEV-infected, apoptotic microglial cells (arrowhead) are also observed. (G) Occasional TUNEL-positive, apoptotic lymphocytes (arrows) are present in the perivascular infiltrates. V: vessel. Indirect peroxidase method (A–E, G), Vectastain Elite ABC-Alkaline Phosphatase Kit (F). DAB (A–G), <i>BCIP</i>/NBT blue (F), Papanicolaou's hematoxylin counterstain. Scale bars: A (left) = 100 µm; A (right), C = 25 µm; B, E = 20 µm; D, F, G = 50 µm.</p

Histopathological changes in the thalamus of a rhesus macaque (No. 2) after intranasal inoculation with JEV.

<p>(A) Non-suppurative encephalitis, represented by moderate, lymphocyte-dominated perivascular infiltration. (B) Small vein with mild perivascular infiltration and activated endothelial cells (arrow). (C) The presence of serum, indicated by staining for von Willebrandt factor, in the parenchyma surrounding vessels with perivascular infiltrates (arrows) indicates marked vessel leakage. (D) Degenerating neuron (arrow) surrounded by glial cells (satellitosis). (E) Microglial nodule with occasional apoptotic cells (black arrow). (F) Staining for GFAP highlights the presence of large numbers of activated astrocytes (reactive astrocytosis). A, B, D, E: Hematoxylin-eosin stain. C, F: Indirect peroxidase method, NovaRed (C), DAB (F), hematoxylin counterstain. Scale bars: A, C, F = 50 µm; B, D, E = 20 µm.</p

Proinflammatory markers in the thalamus of rhesus macaques after intranasal inoculation with JEV (No. 2 (A, B, D–F), No. 11 (C)).

<p>(A) Microglial cells (small arrows), leukocytes in the perivascular infiltrates (arrowheads), perivascular macrophages (large arrow) and astrocytes (inset) express iNOS. (B) Nitrotyrosine expression is observed in microglial cells (arrowheads) and astrocytes (arrows). VL: vessel lumen. (C) MMP-2 expression is diffusely seen in reactive astrocytes. (D) MMP-9 is mainly expressed by neurons. (E) TNF-α (left: brown signal) is expressed by microglial cells (left: arrows; right: arrowheads) that are identified based on their CD68 expression (left: blue signal) and astrocytes (right: arrows). (F) IFN-α expression is seen in astrocytes (left; arrow) and neurons, both unaltered (left: arrowheads; right: arrow) and degenerating (right: arrowhead), as demonstrated in satellitosis. Microglial cells surrounding the neuron are also positive. Indirect peroxidase method (A–F), Vectastain Elite ABC-Alkaline Phosphatase Kit (E, left); DAB (A–F), <i>BCIP</i>/NBT blue (E, left), Papanicolaou's hematoxylin counterstain. Scale bars A–D, F left = 50 µm. E, F right = 20 µm.</p

Animals, JE challenge virus, infectious doses and time of necropsy.

<p>pfu – plaque-forming unit.</p><p>Animals were euthanized at the onset of stupor or coma.</p

Isolation and complete genome analysis of neurotropic dengue virus serotype 3 from the cerebrospinal fluid of an encephalitis patient

<div><p>Although neurological manifestations associated with dengue viruses (DENV) infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3) from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus.</p></div

Mfold RNA structure prediction for the complete 3’UTR of DENV-3 prototype strain H87 (KU050695) and the neurotropic DENV-3 201610225 (KY863456).

<p>Stop codon (UAA) is indicated with red font in both RNA structure just upstream nucleotide 10268. Nucleotide insertion and changes in isolate 201610225 are indicated with yellow and red circles, respectively. The positions of the nucleotide changes are indicated according to 201610225 nucleotide numbering with H87 nucleotide numbering in parentheses. The two stem-loops (SLI and SLII), two dumbbells (DBI and DBII), and small hairpin 3’ stem-loop (SHP-3’SL) structures are shown.</p

Amino acid substitutions between 201610225, DENV-3 prototype strain (H87), and DENV-3 Indonesian historical strain (Sleman/78 and den3_88) genome sequence.

<p>Amino acid substitutions between 201610225, DENV-3 prototype strain (H87), and DENV-3 Indonesian historical strain (Sleman/78 and den3_88) genome sequence.</p

Isolation of DENV-3 from CSF and serum samples of an encephalitis patient in Vero and C6/36 mosquito cell cultures.

<p>Phase contrast light microscopy of uninfected monolayer Vero cells (A); infected Vero cells at day 5 after inoculation with CSF sample (B); and infected Vero cells at day 10 after inoculation with serum sample (C). Indirect immunofluorescent assay (IFA) staining of uninfected C6/36 mosquito cells (D); infected C6/36 mosquito cells at third passage of inoculation with CSF sample (E). For IFA staining, cells were stained with mouse anti-DENV-3 monoclonal antibody (clone 5D4), followed by FITC-conjugated goat anti-mouse IgG. Evans blue was used as a counterstain (red). The arrows in B and C point to cytopathic effect (CPE), while the arrows in E point to DENV-infected cells.</p