Alignment of amino acids 577−740 of rhesus LCV EBNA-3B from LCL8664 cells (middle lines) with the previously published sequence ([<b>21</b>], lower lines), and amino acids 571−768 of EBV AG876 EBNA-3B (top lines).

<p>Numbers indicate amino acid positions of rhesus LCV EBNA-3B and arrows indicate where the sequences of rhesus LCV EBNA-3B diverge and then return to identity.</p

Expression of EBNA-3A and EBNA-3B in transfected and latently infected cells.

<p>(A) Cos cells transfected with pSGrhEBNA3A1 and pSGrhEBNA3A1-del express EBNA-3A beginning at the first methionine without (lane 2) or with (lane 3) a deletion in the putative RBP-Jκ binding site, respectively. Cos cells transfected with pSGrhEBNA3A2 and pSGrhEBNA3A2-del express EBNA-3A beginning at the second methionine without (lane 4) or with (lane 5) a deletion in the putative RBP-Jκ binding site, respectively. Cos cells were transfected with pSGrhEBNA3B and pSGrhEBNA3B-del, with a deletion in the putative RBP-Jκ binding site (lanes 6 and 7, respectively). Cos cells were transfected with empty vector (pSG5, lane 8). (B) Detection of EBNA-3A in Vero cells infected with VRP-EBNA-3A, or in LCL8664 cells and in a rhesus monkey LCL (LCL-V). Arrow indicates location of EBNA-3A. (C) Detection of EBNA-3B in Vero cells infected with VRP-EBNA-3B, or in LCL8664 cells and LCL-V. Arrow indicates location of EBNA-3B. Additional bands noted in cells infected with VRP expressing EBNA-3A or -3B are likely due to overexpression of the protein.</p

Detection of rhesus LCV-specific CD4 and CD8 T cell responses to gp350 or EBNA-3A and EBNA-3B in rhesus monkeys.

<p>Animals were immunized with soluble gp350, VRP-gp350, a combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS before challenge with wild-type virus. Pre indicates PBMCs obtained before vaccination; post indicates PBMCs obtained after vaccination.</p

Detection of antibody to rhesus LCV VCA in monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS.

<p>Detection of antibody to rhesus LCV VCA in monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS.</p

Detection of gp350 in supernatant from cells transfected with a plasmid expressing soluble gp350, lysate from DF-1 cells infected with MVA-gp350, and lysate from Vero cells infected with VRP-gp350.

<p>Supernatant from cells transfected with control plasmid pGL3-GFP, or lysates from cells infected with MVA or VRP-GFP are negative controls.</p

Detection of rhesus LCV DNA in the blood of monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS.

<p>Real time PCR was performed using a probe that detects EBER1 DNA.</p

Detection of gp350 antibody by luciferase immunoprecipitation assay in rhesus monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS before challenge with wild-type virus.

<p>gp350 antibody levels in naturally infected monkeys are shown. Antibody levels are measured as luminometer units. Cut off value is shown as horizontal dotted line, which was determined as the mean +2 standard deviations of the blank signal (open circles). Horizontal bars indicate geometric mean, asterisks indicate p<0.05 (Mann-Whitney's U-test), NS indicates not significant.</p

Detection of rhesus LCV EBER1 in the blood of monkeys immunized with soluble gp350, VRP-gp350, a combination of VRP-350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS.

<p>RNA was isolated from PBMCs and reverse transcription was performed followed by real time PCR with a probe that detects EBER1 DNA.</p

HAstV-NIH infects neurons with the highest burden in the cerebellum and brainstem.

(A—H) Astrovirus capsid protein (brown) in neuronal perikarya and projections (dendrites and axons) in indicated brain structures. Heatmap (yellow-low to red-high) of the neuronal virus infection burden along the neural axis is shown on the left of each image panel. Colors are based on the normalized counts of AstV+ neurons per mm2 of tissue area for all structures, except for Purkinje cells (PCs) in the cerebellar cortex, which is based on the number of AstV+ PC profiles per linear mm of PC layer. (I) Radar graph represents the entire brain clockwise from the cerebral cortex to the medulla and shows the normalized numbers of AstV+ neurons in each structure (data are presented as mean counts [red line] ± SE [gray dashed lines]). (J and K) Double immunofluorescent staining for the neuronal somatodendritic marker MAP2 (red), AstV capsid protein (green), and DAPI nuclear counterstain (blue) in the indicated brainstem structures with high neuronal infection burden. Note: (i) accumulation of aging pigment lipofuscin, which is known to be autofluorescent, produces an intense red signal in the perikarya of AstV-negative (AstV-) MAP2++ neurons and dendritic profiles and this signal colocalizes (yellow) with AstV (green) signal in infected neurons; and (ii) there is a paucity of AstV-/MAP2++ dendritic profiles and MAP2 signals are either low (MAP2+) or absent (MAP2-) in AstV+ dendritic profiles. Labeling keys used in (J and K) are provided at the bottom of the figure. Scale bars: 10 μm.</p

Detection of astrovirus in the brain of an immunocompromised patient with an encephalitis.

(A) Fold change relative to control for probes to virus families hybridizing to RNA from the brain of the patient with AstV-NIH. (B) Detection of the astrovirus RNA in the brain of the patient with AstV-NIH by PCR. (C and D) In situ hybridization signals (magenta-red) in indicated brain tissue samples using the negative strand astrovirus RNA probe (upper panels) or RNA probes for actin as controls (lower panels). (PDF)</p