Epstein-Barr virus infection is not a characteristic feature of multiple sclerosis brain

Abstract

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) that is thought to be caused by a combination of genetic and environmental factors. To date, considerable evidence has associated Epstein-Barr virus (EBV) infection with disease development. However, it remains controversial whether EBV infects multiple sclerosis brain and contributes directly to CNS immunopathology. To assess whether EBV infection is a characteristic feature of multiple sclerosis brain, a large cohort of multiple sclerosis specimens containing white matter lesions (nine adult and three paediatric cases) with a heterogeneous B cell infiltrate and a second cohort of multiple sclerosis specimens (12 cases) that included B cell infiltration within the meninges and parenchymal B cell aggregates, were examined for EBV infection using multiple methodologies including in situ hybridization, immunohistochemistry and two independent real-time polymerase chain reaction (PCR) methodologies that detect genomic EBV or the abundant EBV encoded RNA (EBER) 1, respectively. We report that EBV could not be detected in any of the multiple sclerosis specimens containing white matter lesions by any of the methods employed, yet EBV was readily detectable in multiple Epstein-Barr virus-positive control tissues including several CNS lymphomas. Furthermore, EBV was not detected in our second cohort of multiple sclerosis specimens by in situ hybridization. However, our real-time PCR methodologies, which were capable of detecting very few EBV infected cells, detected EBV at low levels in only 2 of the 12 multiple sclerosis meningeal specimens examined. Our finding that CNS EBV infection was rare in multiple sclerosis brain indicates that EBV infection is unlikely to contribute directly to multiple sclerosis brain pathology in the vast majority of cases.

Figure 1

ISH revealed EBER⁺ cells were not present in multiple sclerosis brain. LFB staining identified regions of considerable demyelination (plaques) in representative multiple sclerosis tissue specimens obtained from patients multiple sclerosis_1 (A and D) and _2 (G); blue = positive staining for myelin; pink = negative for myelin. Immunohistochemistry identified a prominent CD20⁺ B cell infiltrate (B and E) in adjacent sections within demyelinated areas (brown-positive staining for antigen) in some specimens and a sparse CD20⁺ B cell infiltrate in others (H). ISH revealed no EBER⁺ cells within demyelinated regions (C, F and I). EBER was readily detected (brown nuclear staining) in two EBV-positive CNS lymphomas (J and K). Figures A, D and G (100×); B, C, E and F (200X); H and I (400×); J, K and insets (1000×). (L) ISH revealed no EBER⁺ in any of the 23 multiple sclerosis specimens examined obtained from 12 autopsy cases, yet all specimens had a detectable CD20⁺ B cell infiltrate. Each dot represents the total number of positive cells counted in five 200× fields. The bar represents mean cell number observed over the 23 multiple sclerosis specimens examined.

Figure 2

Quantitative real-time PCR could detect genomic EBV and EBER1 in a single EBV infected cell. The EBV-positive lymphoblastoid (IB4) cell line was sorted (100 cells to 1 cell) into a 96-well plate and real-time PCR performed (in triplicate) as described in Methods section. Genomic EBV W repeats (A), EBER1 (B) and CD20 (B) were detectable in a single EBV-positive cell. Actin (A) and B2M (B) served as controls. Genomic EBV (C) and EBER1 (D) were also readily detectable in an EBV-positive post-transplant B cell lymphoma and not in an EBV-negative B cell lymphoma. Real-time PCR was performed for 50 cycles, and values shown indicate 50-Ct.

Figure 3

EBV was not detectable by quantitative real-time PCR in multiple sclerosis specimens with a confirmed B cell infiltrate. Multiple sclerosis specimens containing white matter lesions were serially sectioned for alternate DNA or RNA isolation (A; processed under same conditions used for post-transplant B cell lymphomas). Genomic EBV (B) or EBER1 (C) was not detectable by real-time PCR (performed in triplicate) in all multiple sclerosis specimens examined. All multiple sclerosis specimens contained a detectable CD20⁺ B cell infiltrate (C). Few EBV-positive cells (IB4) could be detected when processed with a multiple sclerosis tissue specimen for DNA (two cell sensitivity; D) or RNA (one cell sensitivity; E). Actin (B and D) and B2M (C and E) served as controls for real-time PCR. Each data point represents the mean value (50-Ct) over the four samples processed for DNA or RNA.

Figure 4

EBV was detected in very few multiple sclerosis specimens containing B cell infiltration within the meninges and parenchymal B cell aggregates. Fixed-frozen tissue specimens were sectioned with minimal waste and stained with LFB, CD20 and EBER. LFB staining identified B cell aggregates in regions of considerable demyelination (A and D—Multiple sclerosis_UK10) (blue = positive staining for myelin; pink = negative for myelin). Dense CD20⁺ B cell aggregates are shown in the brain parenchyma in one case (B and E—Multiple sclerosis_UK10), with loose B cell infiltrates identified within the meninges (G) of several others (H and not shown), while most cases had a diffuse B cell infiltrate. ISH revealed no EBER⁺ in all 12 multiple sclerosis specimens examined (C, F and I and not shown). An EBV-positive CNS lymphoma (I inset) served as a positive control for EBER ISH. Snap-frozen multiple sclerosis tissue specimens from the same cases were serially sectioned (10 μm sections; see Methods section) for alternate DNA or RNA isolation. Genomic EBV (J) was detected in 2 of 12 multiple sclerosis samples examined while EBER1 (K) was detected in one sample with detectable genomic EBV. All samples had a detectable CD20-positive B cell infiltrate (K). Note the sample with detectable genomic EBV but not EBER1 only contained EBV within one of two tissue sections examined (value reported is the average seen within the positive section only). Figures A and D (200×); B, C, E, F and G (400×); H, I and inset (1000×).