Molecular signature of Epstein-Barr virus infection in MS brain lesions

CD20⁺ B lymphocytes (A–C) and CD138⁺ plasma cells (black arrowheads) (D–F) in the parenchyma and vasculature in a chronic active plaque (CAP) (A and D), in a chronic plaque (CP) (B and E), and in the vasculature of a healthy control brain sample (C and F). Percentage of CAP, CP, and control brain samples expressing CD138 protein detectable by immunohistochemistry are shown (M). We observed latent membrane protein-1 (LMP-1) expression (red arrowheads) in CAP, CP, and control brain samples (G–I). Percentage of CAP, CP, and control brain samples expressing LMP-1 protein detectable by immunohistochemistry are shown (N). Cells expressing LMP-1 (red arrowheads) were found in the vasculature of CAPs (G) and control brains (I), and within the parenchyma in a CP (H). The expression of the viral immediate-early protein BZLF1 was not observed in a CAP (J) and was observed in and around the vasculature in tissue from a CP (red arrowheads) (K). BZLF1 was also observed to a lesser extent in healthy controls (red arrowheads) (L). Percentage of CAP, CP, and control brain samples expressing BZLF1 protein detectable by immunohistochemistry are shown (O). Scale bar = 50 μm. Pictures are representative of analysis from 17 MS samples and 9 healthy controls. EBV = Epstein-Barr virus.

Pictures are representative of results from 2 different experiments using EBV-positive (tonsil with infectious mononucleosis) and EBV-negative (normal tonsil) control samples. Immunohistochemistry revealed the presence of CD20⁺ B lymphocytes (A–B) and CD138⁺ plasma cells (C–D) in our positive and negative control tissue (A–D). LMP-1⁺ (brown staining) and BZLF1⁺ (brown staining emphasized with black arrows) cells are shown in a tonsil from a patient with infectious mononucleosis (E, G). No LMP-1⁺ or BZLF1⁺ cells were observed in our negative control tissue (normal tonsil) (F, H). Scale bar = 50 μm.

In situ hybridization for EBER detects EBER⁺ cells (blue-black nuclei, black arrows) in 4 representative MS brains (B, D, H, and K) and 1 representative control brain (F). In situ hybridization for EBER and immunohistochemistry for LMP-1 show EBER⁺ and LMP-1⁺ cells in the same region or nearby regions in 2 representative MS brains (G–L). Tonsil tissue from a patient with infectious mononucleosis (M–O) shows colocalization of EBER and LMP-1. RNA preservation in samples is corroborated by in situ hybridization for oligo dT in serial sections from the same MS and control samples (A, C, E, G, J, and M). Scale bar = 50 μm.