Wednesday, 9 April 2014

Grand Challenges in MS (9): EBV genomes

Is the strain of EBV that causes MS a teenage mutant ninja turtle? #MSBlog #MSResearch

"At last big genomics comes to EBV. Not all EBV viruses are the same; within the populations of the world different strains of EBV exist and within an individual different forms of EBV can evolve. The rapid evolution of viruses between populations and within individuals is well described for many viruses so why should EBV be any different? Why is this important? I hypothesised several years ago that maybe the strain of EBV that triggers, or drives, MS is a different strain to that that lives and thrives in the  general population. In other words for EBV to cause MS it needs to be a mutant-EBV. There are analogies to this hypothesis. Almost all the common viruses that result in chronic slow viral infections of the brain have mutant strains that do the dirty work. This was first described for the measles virus; the strain of measles that causes subacute sclerosing panencephalitis (SSPE); the measle virus that causes SSPE has a mutation in its M protein. Similarly, the strain of herpes simplex virus that causes encephalitis has a specific mutation in its genome that allows it infect the brain. JCV that causes PML in MSers on natalizumab has several mutations in its coat protein, VP1, and a mutation in the part of its genome that regulates its function. These examples support the hypothesis of why we should be taking a deep look into the EBV genome of MSers. Where would we look? The obvious place to look is in the blood or saliva, where the virus is found normally. However, this may the wrong compartment to look. I would suggest we  take a look in the brain and cervical, or neck, lymph nodes of people with MS. This is the place we are  more likely to find the mutant strain lurking. What is needed to do these studies? (1) Tissue collected in a special way from people dying with MS, (2) a new breed of scientist and (3) deep pockets."

"Tissue. When I asked the brain bank to collect deep cervical lymph nodes, spleen and salivary glands from MSers donating their brain I was told that the current consent form did not cover this level of tissue donation. I would have to set-up a special donation process. As this was only a hypothesis and I did not have the resources I couldn't implement this study. if I submitted a grant to get this study funded  the obvious question the reviewer's would ask is the EBV virus in the brain of MSers? That is a very contentious issue and for those of you have been following the field will have seen that the answer is not clear. One group in Italy seems to find EBV with ease (see Serafini  et al. abstract below), whereas several other groups could not reproduce the finding (Lassmann et al. below). Ute Meier in our group has found EBV in the brain, but at a much lower level (Tzartos et al below). So more work needs to be done. Would grant peer-reviewer's give us the thumbs up? I suspect not."

"A new breed of scientist. To test the tissue-specific EBV strain hypothesis we need virologists with skills in deep sequencing and bioinformatics. These people exist but not in the field of MS. if anyone out there with the skills is keen to bring their know-how and expertise the field of MS please contact me I would love to support you. Unfortunately, I don't have the skill set to take this project on; any grant review panel would reject an application from me without letting me pass GO."

"Deep pockets. As always research costs money and this project will require a team of people with funding for 3-5 years to make any dent in this hypothesis. The project may have to involve technologies that push the bubble of technological innovation. For example, I envisage having to capture single cells from  the brains of MSers and sequence the EBV genomes from individual cells. All this will requires time, patience, access to emerging technology, skills and money."

"Can we afford not to do these sort of projects? No. If all the evidence points towards EBV being the cause of MS we need to find a mechanism to explain how EBV causes MS. Ideas and hypotheses about how EBV causes MS are a dime a dozen. What we need are experiments to disprove the increasingly long list of hypotheses."

Is the strain of EBV that causes MS a mutant ninja turtle?

Epub: Santpere et al. Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1000 Genomes Project. Genome Biol Evol. 2014 Mar.

Background: Most people in the world (~90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B-cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. 


Aim: To perform a detailed comparative genomics analysis of EBV including, for the first time, EBV strains derived from healthy individuals we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. 

Methods: Since strain B95-8 was used to transform B-cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. 

Results: After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605) we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. 

Conclusion: We found that latency genes harbour more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite co-evolution.

Do we find EBV in the brains of MSers? YES


Serafini et al. Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J Exp Med. 2007 Nov 26;204(12):2899-912.

Contrary to previous studies, we found evidence of EBV infection in a substantial proportion of brain-infiltrating B cells and plasma cells in nearly 100% of the MS cases examined (21 of 22), but not in other inflammatory neurological diseases. Ectopic B cell follicles forming in the cerebral meninges of some cases with secondary progressive MS were identified as major sites of EBV persistence. Expression of viral latent proteins was regularly observed in MS brains, whereas viral reactivation appeared restricted to ectopic B cell follicles and acute lesions. Activation of CD8+ T cells with signs of cytotoxicity toward plasma cells was also noted at sites of major accumulations of EBV-infected cells. Whether homing of EBV-infected B cells to the CNS is a primary event in MS development or the consequence of a still unknown disease-related process, we interpret these findings as evidence that EBV persistence and reactivation in the CNS play an important role in MS immunopathology.

Do we find EBV in the brains of MSers? NO

Recent epidemiological and immunological studies provide evidence for an association between Epstein-Barr virus infection and multiple sclerosis, suggesting a role of Epstein-Barr virus infection in disease induction and pathogenesis. A key question in this context is whether Epstein-Barr virus-infected B lymphocytes are present within the central nervous system and the lesions of patients with multiple sclerosis. Previous studies on this topic provided highly controversial results, showing Epstein-Barr virus reactivity in B cells in the vast majority of multiple sclerosis cases and lesions, or only exceptional Epstein-Barr virus-positive B cells in rare cases. In an attempt to explain the reasons for these divergent results, a workshop was organized under the umbrella of the European Union FP6 NeuroproMiSe project, the outcome of which is presented here. This report summarizes the current knowledge of Epstein-Barr virus biology and shows that Epstein-Barr virus infection is highly complex. There are still major controversies, how to unequivocally identify Epstein-Barr virus infection in pathological tissues, particularly in situations other than Epstein-Barr virus-driven lymphomas or acute Epstein-Barr virus infections. It further highlights that unequivocal proof of Epstein-Barr virus infection in multiple sclerosis lesions is still lacking, due to issues related to the sensitivity and specificity of the detection methods.

Do we find EBV in the brains of MSers? YES, but ....


Tzartos et al. Association of innate immune activation with latent Epstein-Barr virus in active MS lesions. Neurology. 2012 Jan 3;78(1):15-23. 

OBJECTIVE: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in themultiple sclerosis (MS) brain.

METHODS: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization.

RESULTS: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro.

CONCLUSION: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

5 comments:

  1. I believe EBV infects B cells unlike JCV which infects white matter. So, if this is a mutation if the normal EBV it would have to have significant differences than the normal EBV such that it infects the white matter. My question would be is this characteristic of a mutated virus, or do they need some commonality to be considered the same virus family?

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  2. Talk to Prof Compston, the man is a genius.

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  3. EBV has the ability to infect many cells that are not part of the immune system for example muscles, epithelial cells and is known to cause nasal cancers and the nose is closely linked to the brain.

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  4. Many aspects of EBV, EBVinfection as well as and reaction to EBV have been linked to MS.
    - anti viral effects of IFN beta
    - 'homing'of EBV in B-cells
    - effects of anti CD20 MoAbs in MS
    - 'standard' IgG titer angaints 'EBV'' does not protect against (all cases of) overt MS. Different EBV strains might be involved.

    In order to address any search for a therapeutical approach: What are the pathogenic as well as the immunogenic peculiarities of these pathogenic straisn of EBV? and what are the EBV-epitopes that resemble those presented by (micro-)glial and astrocytes?
    - is inducton of anergy with regard to these 'similar' a reasoable approach, or should one focus on overall (more blunt) therapies that could be generalized (& commercialized)?

    Please comment!
    George

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