However, in cases where considerable cellular content was present within the meninges, occasional EBER positive cells were detected. Interestingly, cases which were EBER-ISH positive in meninges, were almost always positive in white matter as well, but not vice versa. The presence of B-cell follicles is thought to be linked to progressive MS and cases with prominent subpial grey matter pathology [ 45 , 47 , 60 ]. Due to lack of clinical data, we cannot rule out the possibility that most of our cases were not progressive disease, hence the lack of meningeal infiltration.
Only occasional BZLF1 positive cells were observed. In general, EBER positive cells had lymphocyte-like morphology. Occasionally, positive cells were noted which did not have the typical lymphocyte appearance. These observations were consistent with the findings reported in the original autopsy histologic examination of these cases. In one of these cases, considerable level of expression of these cellular markers was noted. This case contained prominent CD20 positive perivascular cuffing with EBER positive cells distributed in and near to the inflamed blood vessels.
We performed double staining on 18 EBV heavily infected cases. Double staining for EBV EBER-in situ hybridization: dark blue staining and different cellular markers immunohistochemistry: brown in the white matter of 3 different heavily infected MS cases. The pattern of double-staining seen in these 3 cases is representative of that seen in other double-positive cases.
Double positive cells are indicated by the arrows. However, the link between EBV and MS is not universally accepted since some studies have failed to consistently find the virus in MS brains [ 37 , 43 ]. It has been argued that these conflicting results are probably due to differences in the tissue samples examined and the sensitivity and specificity of the techniques used [ 6 ]. We believe that the extent and degree to which tissues are examined for the presence of EBV is also an important contributing factor to the differences observed.
Consistent with some previous reports, we also observed evidence of EBER positive cells in an MS case where perivascular regions were infiltrated with CD20 positive B-cells [ 46 , 61 ]. Previous reports have indicated that the presence of B cell-like follicles is a common occurrence in MS lesions and meninges, and could be the reservoir for EBV in the inflamed CNS [ 38 , 47 , 62 ].
These follicles may be associated with subpial demyelination, cortical atrophy and increased activation of microglia leading to grey matter injury [ 56 , 64 , 65 ]. However, the meninges in most of our cases did not have any prominent lymphoid infiltration. It is possible that some of these discrepancies are due to the heterogeneous dissemination of meningeal immune infiltrates and ectopic B cell follicles [ 59 , 60 , 66 ].
These tertiary structures are thought to be dependent upon disease length and subtype [ 47 , 64 ]. Differences in the observations, could also be due to variations in sampling and tissue processing.
In our hands, using this technique we have been able to identify a single EBV positive cell in a section [ 54 , 55 , 72 ]. Despite this level of infection, the direct demonstration of EBV infected cells in the brain of MS cases is significant and could imply a potential role for EBV in the pathogenesis of the disease.
The findings in this comprehensive study support a number of previous reports indicating that EBV is present in a significant proportion of MS cases [ 45 — 47 ]. Although in our study we did not find distinct EBV clusters or follicles, we did however find that the virus was transcriptionally active in the infected cells. In contrast to our study and those mentioned above, some reports have failed to find the presence of EBV in MS brains [ 37 , 44 , 73 ].
Although EBV is a highly B-cell tropic virus, our findings suggest that in brain tissues of MS cases, cells other than B-cells can also be infected. The amoebic form taken by virtually all microglia seen in our cases indicated that these cells were in an activated state. Active microglia were shown recently to stimulate a subtype of astrocytes that can be toxic to neurons and myelin-producing oligodendrocytes [ 74 ]. Where EBV fits in this inflammatory cycle has to be investigated.
Also, how EBV enters microglia and astrocytes is unknown. Thus, occasional uptake of EBERs by neighboring cells, such as microglia and astrocytes, could take place in the absence of bonafide viral infection [ 77 ].
Additionally, astrocytes in MS have been reported to express BAFF, which prolongs the survival of a subset of infiltrating B cells [ 79 ]. A recent study in marmoset model of experimental autoimmune encephalomyelitis EAE suggested that EBV infection of B cells disrupts the homeostatic cell-to-cell communication that normally occurs between B and T cells, polarizing T cells towards the self-specific inflammatory phenotype [ 8 ].
Our study was not designed to examine the possible mechanisms by which EBV infection may contribute to the demyelination, neuroinflammation and neurotoxicity associated with MS.
Thus further studies are needed to look at how EBV infection may link to these pathological hallmarks of MS. Thus, without meticulous and thorough examination, low level of EBV positivity could be easily missed, leading to underestimation of EBV positivity in MS. Our data also suggests that EBV may infect more than one cell type in MS, including microglia and astrocytes.
However these findings need to be verified and the possible link between the presence of EBV, neuroinflammation, and neurodegeneration remains to be investigated. Age is in years, unless otherwise stated in the table. Numerous EBER-positive cells can be seen scattered in the section using antisense probe A , but not with sense probe negative control B. We would like to thank Ms Pretty Philip for her help and general advice in laboratory techniques. We would also like to thank the National MS Society and the Rocky Mountain MS Centre Tissue Bank for their invaluable assistance and help in providing the brain samples and collecting relevant reports.
National Center for Biotechnology Information , U. PLoS One. Published online Feb 2. John R. Luwen Zhang, Editor. Author information Article notes Copyright and License information Disclaimer. Received Jun 3; Accepted Jan This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. S2 Table: Summary of clinical characteristics of MS cases. Occasional, but very clearly positive cells were seen scattered in the section. S3 Fig: Immunohistochemistry staining for CD Introduction Over 2 million people worldwide suffer from multiple sclerosis MS , a debilitating neurological disease of autoimmune nature [ 1 ].
Table 1 Sequences of the PCR primers used in this study. Open in a separate window. Fig 1. Fig 2. PCR amplification for 3 common herpesviruses. Fig 3.
Fig 4. Fig 5. Immunohistochemistry for EBV proteins. Fig 6. Double staining for EBV and cellular markers. PDF Click here for additional data file. S2 Table Summary of clinical characteristics of MS cases. S3 Fig Immunohistochemistry staining for CD Acknowledgments We would like to thank Ms Pretty Philip for her help and general advice in laboratory techniques. Data Availability All relevant data are within the paper and its Supporting Information files.
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The increased antibody response to Epstein-Barr virus in multiple sclerosis is restricted to selected virus proteins.
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Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents References. Does Epstein—Barr virus infection in the brain drive the development of multiple sclerosis?
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Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology. Epstein-Barr virus latent membrane protein 2A is a B-cell receptor mimic and essential for B-cell survival. As for the one case of MS in someone who remained negative for Epstein-Barr, it is possible that person was infected after the sample was taken, but it is also true that, in diseases that are clinically defined by their symptoms, such as MS, it is highly unlikely that percent of cases derive from the same cause, even if most do, Ascherio says.
But to be sure Epstein-Barr was the culprit, Ascherio and his colleagues also measured antibodies against cytomegalovirus, another herpesvirus, and found no difference in levels in those who developed MS and those who did not. Using a subset of 30 MS cases and 30 controls, they conducted a scan to detect antibody responses to most of the viruses that infect humans.
Again, there was no difference. And to rule out the possibility that infection with Epstein-Barr preceded MS and not the other way around, the team also measured levels of a protein that is elevated in serum when neurons are injured or die and that therefore serves as a marker of the beginning of the pathological process before clinical symptoms appear.
The protein levels only rose after Epstein-Barr infection. One major question remains, however: How does the virus lead to the disease? They proposed several possibilities, such as inducing an autoimmune reaction. Even if Epstein-Barr is the triggering event for MS, infection alone is insufficient for an actual diagnosis.
Epstein-Barr, it appears, has to combine with a genetic predisposition and possibly environmental factors, such as smoking and vitamin D deficiency, to increase risk. Understanding the underlying mechanism will be important, the experts say. Historically, we have thought of MS as an autoimmune disease of unknown etiology. Antivirals that target EBV in infected B cells are one possibility. One of the more exciting developments in MS in recent years was the success of B-cell-depletion therapies.
In earlier work, Hauser and his colleagues found that the tissue damage in MS is primarily directed by B cells, which attack the myelin sheath protecting nerves. The therapies now approved for use are monoclonal antibodies that kill those B cells, thereby easing inflammation.
They are not a cure but are highly effective against MS relapses, reducing the development of new lesions measured by magnetic resonance imaging MRI of the brain by an astounding 99 percent. They are also the only therapies shown to be effective against primary progressive MS, a previously untreatable form of the disease.
Others are already working on vaccines that could prevent infection with Epstein-Barr.
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