After decades of research, a large body of evidence has emerged to support a role for EBV infection in multiple sclerosis disease development. However, precisely how EBV may contribute to multiple sclerosis pathology remains unknown. Early studies using ISH to detect EBV infection in multiple sclerosis brain yielded negative (Hilton et al.
) or inconclusive (Opsahl and Kennedy, 2007
) results. However, recent work has challenged these findings (Serafini et al.
), citing that a failure to detect EBV in multiple sclerosis brain may have resulted from poor tissue preservation, analysis of multiple sclerosis material without relevant inflammatory infiltrates containing B cells or methodological differences. In their study, Serafini and colleagues (2007
) reported that the EBV-associated RNA EBER and EBV protein products were, in fact, present in a large percentage of infiltrating B lymphocytes (40%–90%) in >95% of multiple sclerosis brain cases examined, yet were absent from other cases of inflammatory neurological disease. Furthermore, the authors suggested that EBV infection may be a prerequisite for multiple sclerosis and that the dysregulated EBV infection observed in multiple sclerosis patients may result in a chronic inflammatory state that triggers disease. If true, these results would have significant therapeutic implications for multiple sclerosis as they suggest that vaccination against EBV or regulation of the existing infection may prevent or eliminate disease.
Given the potential therapeutic importance of EBV infection in the CNS and the conflicting reports over its presence in multiple sclerosis brain, we investigated whether EBV infection was a characteristic feature of multiple sclerosis brain using multiple methodologies including ISH, immunohistochemistry and two highly sensitive PCR methodologies that can detect a very low number of EBV-infected B cells. Initially, we characterized the immune cell infiltrate in a large number of multiple sclerosis specimens in order to select a cohort of multiple sclerosis lesions that had a heterogeneous B cell infiltrate and thus had the potential to harbour EBV. From this analysis, a total of 23 multiple sclerosis tissue specimens from 12 autopsy cases were selected from both adult and paediatric multiple sclerosis cases for further analysis. However, while all multiple sclerosis specimens containing white matter lesions examined possessed a heterogeneous B cell infiltrate (B, E, H and L), EBV was not detectable by ISH (). These results were supported by the absence of EBV protein expression in a subset of specimens examined by immunohistochemistry (LMP1 and EBNA2; Supplementary Fig. S3
). In addition, it should be stressed that the absence of EBV in the multiple sclerosis tissue specimens examined is unlikely to reflect sample size as Serafini et al.
) reported that EBV infection was present in >95% of multiple sclerosis cases examined.
Our inability to detect EBV by ISH or immunohistochemistry indicated that EBV infection in these samples was most likely absent or alternatively, present at low levels that may have been missed using the above technologies on single sections of multiple sclerosis lesions. Consequently, we employed two highly sensitive real-time PCR methodologies for the detection of EBV in multiple sclerosis specimens from multiple cases that were shown to have a B cell infiltrate. Two independent quantitative real-time PCR protocols were used for the detection of genomic EBV or EBER1, with the latter expressed at high levels in cells latently infected with EBV. Together with EBER ISH, these approaches are the ‘gold standard’ for EBV detection. However, these real-time PCR methodologies have the added advantage that the PCR products can be sequenced to provide definitive evidence for EBV infection. Seventeen multiple sclerosis lesions from five autopsy cases were extensively sectioned for DNA or RNA isolation (). However, consistent with our ISH results, no EBV was detected, despite the fact that all lesions harboured a B cell infiltrate () and that our assays were extremely sensitive, in that as few as two EBV-positive cells provided a positive signal ().
In the recent study by Serafini and colleagues (2007
), it was reported that ectopic B cell follicles within the meninges were the main site of EBV persistence, although EBV was found within almost all multiple sclerosis specimens with relevant inflammatory material, whether follicles were present or not. Our observation that EBV was not detectable in our cohort of multiple sclerosis white matter lesions that harboured a B cell infiltrate led us to assess whether EBV infection was restricted to multiple sclerosis tissue specimens containing B cell infiltration within the meninges and parenchyma (). To address this, we examined 12 multiple sclerosis specimens (12 fixed-frozen and 12 snap-frozen blocks), which were obtained from the same tissue bank as those used in the Serafini study where the EBV positivity rate was reported to be >95%. These specimens represented adjacent tissue blocks from some (not all) of the same cases. Consistent with this previous study, large B cell aggregates were identified in a subset of cases predominantly within the brain parenchyma, with loose B cell infiltrates found in the meninges in a subset of cases (). In contrast to the prior study (Serafini et al.
), EBV was not detectable by ISH in all cases examined while EBV was detectable in our positive control (I inset). Consistent with our ISH data, EBV was absent in 10 of the 12 matching frozen cases examined by real-time PCR. Genomic EBV and EBER1 were detected in only one case. In the other case, only genomic EBV was detected in one section, despite the fact that B cells were detected in both sections examined. These combined data demonstrate that while we could, as expected, occasionally detect the low levels of EBV signals in human tissue, EBV was largely absent in multiple sclerosis brain.
Based on the seroepidemiological evidence obtained over several decades, a growing number of studies have shown an association between EBV and multiple sclerosis. These findings include but are not limited to; the higher incidence of EBV infection in both adult and paediatric multiple sclerosis cases relative to controls (Bray et al.
; Sumaya et al.
; Wandinger et al.
; Alotaibi et al.
; Haahr and Hollsberg, 2006
; Pohl et al.
; Ascherio and Munger, 2007
); and the increased numbers of EBV reactive CD8 (Hollsberg et al.
; Cepok et al.
; Lunemann et al.
; Jilek et al.
) and CD4 T cells (Lunemann et al.
) in the periphery of multiple sclerosis patients. However, in the absence of increased viral levels in the serum of multiple sclerosis patients (Wagner et al.
; Lunemann et al.
), or significant infection levels within the CSF of multiple sclerosis patients (Alvarez-Lafuente et al.
), and our finding that EBV was largely absent from multiple sclerosis brain, the contribution EBV may make to multiple sclerosis development remains unknown.
While the seroepidemiology studies have demonstrated a clear association between EBV infection and multiple sclerosis, it must be stressed that caution should be taken in interpreting these results in terms of a causal relationship. This is because it remains possible that the observed association is a consequence of host factors that predispose individuals to both multiple sclerosis and infection with certain viruses such as EBV (Niller et al.
). Furthermore, many studies have reported increased antibody titres to a range of pathogens in multiple sclerosis patients including EBV, measles virus (Panelius et al.
; Shirodaria et al.
), rubella virus (Shirodaria et al.
) and Chlamydia pneumoniae
(Sriram et al.
) as evidence for potential involvement in disease development. However, the involvement of many of these pathogens has been called into question, as vaccination against measles, mumps and rubella has not altered the incidence of multiple sclerosis.
In addition to its association with multiple sclerosis, epidemiological studies also support an association between EBV infection and systemic lupus erythematosus (James et al.
). Furthermore, EBV infection has also been implicated in a number of other autoimmune conditions including, but not limited to, rheumatoid arthritis (RA), Sjogrens syndrome and autoimmune thyroiditis (reviewed in Niller et al.
). Not surprisingly, the implicated diseases are thought to include a B cell-mediated role in their immunopathology and like the situation in multiple sclerosis, the per cent of these patients that have been infected by EBV exceeds that of the general population. However, no direct role for EBV infection in the immunopathology in any autoimmune disease has been demonstrated. The suspected causal role of autoimmunity by EBV presumably began when antibodies directed toward the virus were shown to be elevated in patients with systemic lupus erythematosus (Evans et al.
). Since then a number of autoantibodies reactive with both auto-antigens and EBV components, arising presumably through molecular mimicry, have been found in RA and systemic lupus erythematosus and multiple sclerosis (Niller et al.
). These and other findings have continued to support the notion of an EBV-associated causative role in autoimmunity. An example of direct evidence for such an autoimmune disease-propagating event would show that pre-existing auto-reactive B cells become immortalized through EBV infection and are protected against mechanisms of peripheral tolerance such as anergy and thus go on to produce disease-associated autoantibodies. It is this mechanism that has been proposed to play a role in the immunopathology of RA (Pender, 2003
). Investigation into the presence of EBV in the RA synovium, the site in which immune-mediated pathogenesis is observed, indicated EBER RNA was detectable in 8 of 34 specimens examined (Takei et al.
). Subsequent studies failed to confirm these findings (Alspaugh et al.
; Fox et al.
), indicating that a causal role for EBV infection in the pathology of RA is indirect if present at all. Our investigation into the role EBV plays at the site of injury in multiple sclerosis, the CNS, has led us to the same conclusion.
In summary, despite an exhaustive search using multiple methodologies we have shown that EBV appears largely absent from multiple sclerosis brain. While our findings do not exclude the notion that EBV may contribute to multiple sclerosis via an indirect effect on immune function or through molecular mimicry between EBV and CNS antigens, our results lead us to conclude that EBV infection is unlikely to contribute directly to multiple sclerosis immunopathology in the vast majority of cases.