Epstein Barr Virus the Cause of Multiple Sclerosis

Although many studies have found a kind of a relationship between an Epstein-Barr Virus (EBV) and the development of Multiple Sclerosis (MS), a fundamental aspect of this relationship remains uncertain. What is the cause of Multiple Sclerosis (MS)? In this study, we re-analysed the data as published by Wandinger et al. and were able to establish a new insight: without an Epstein-Barr Virus (EBV) infection no development of Multiple Sclerosis (MS). Furthermore, we determined a highly significant causal relationship between Epstein-Barr Virus (EBV) and multiple sclerosis. Altogether, Epstein-Barr Virus (EBV) is the cause of multiple sclerosis (p-value 0.0004251570).


INTRODUCTION
Multiple sclerosis is one of the most common inflammatory demyelinating diseases of the central nervous system, affecting people of almost all ages in many parts of the world. MS affects more than 2 million 1 people worldwide and is driven by a pathological inflammation. The first description of multiple sclerosis (MS) dates back to the 14th century 2 , but it was Jean-Martin Charcot (1825Charcot ( -1893, the father of neurology 2 who provided the first detailed description of MS in 1868 (described as "la sclérose en plaques" 3 ). The aetiology of Multiple sclerosis is still not well understood even if MS is not directly inherited. Some environmental factors such as latitude, vitamin D, or cigarette smoking 4 and other are unlikely to explain the cause of multiple sclerosis. Epidemiological studies [5][6][7] reported some evidence that EBV might be involved in the pathogenesis of MS. EBV itself is a member of the herpes family of viruses and persists after a primary infection latently in resting memory B cells [8][9] during the lifetime of the host. The prevalence of IgG antibodies to varicella zoster virus (VZV), herpes simplex virus (HSV), and cytomegalovirus (CMV) did not differ between multiple sclerosis cases and controls 10 . Numerous studies investigated the relationship between Epstein-Barr virus (EBV) and multiple sclerosis (MS) and provided some evidence that the titres of EBV antibodies are significantly lower among seropositive controls when compared with sero-positive MS cases 11-12 . In line with observations like these, recent systematic reviews [13][14] provided evidence of an association between MS and sero-positivity for different EBV antibodies but failed to provide any etiological link between EBV and the pathogenesis of MS. Only one study was able to provide evidence of a causal relationship 15 between Epstein-Barr virus and multiple sclerosis. Still, the relationship between Epstein-Barr virus and multiple sclerosis remains a matter of controversy.

MATERIAL AND METHODS
Multiple sclerosis is very heterogeneous in nature and symptomatology and severity is varying greatly from patient to patient. Patient may present with a wide variety of clinical symptomatology including sensory, visual, motor, cerebellar and brainstem dysfunction. MS can restrict the individual's income-earning ability, resulting in a major financial burden on the society, the health system, the family and the patient. Considering the costs associated with MS disease severity, non-pharmaceutical or pharmaceutical interventions aimed at delaying the progression of disease may help to reduce the burden of MS.

Search strategy
For the questions addressed in this paper, Pubmed was searched for case-control studies conducted in any country which investigated the relationship between Epstein-Barr virus and MS. The search in PubMed was performed while using medical key words like "case control study" and "Epstein-Barr virus" and "multiple sclerosis" and "PCR DNA" et cetera. More than 600 articles were identified from which from which more than 30 published studies were selected for a reanalysis. The abstracts of the articles found where saved as a *.txt file while using PubMed's support (Menu: Send to, Choose Radio Button: File, Choose Format: Abstract (text). Click bottom "create file"). The created *.txt file was converted into a *.pdf file. If necessary, the original article was studied. The abstracts where studied within the *.pdf file. Those articles were considered for a review which provided access to data without any data access barrier; no data access restrictions were accepted. Additionally, references from relevant publications and review articles were checked. Studies were excluded if insufficient data were provided to calculate the measures of relationship or if there were data access barriers.

Statistical analysis
All statistical analyses were performed with Microsoft Excel version 14.0.7166.5000 (32-Bit) software (Microsoft GmbH, Munich, Germany). In order to simplify the understanding of this article, to increase the transparency for the reader and to correct some of the misprints of former publications, several of the following lines are repeated word by word and taken from former publications.

The 2x2 Table
The meaning of the abbreviations a t , b t , c t , d t , N t of the data table used are explained by a 2 by 2- table Table 1. In general it is (a t +b t ) = A t, (c t +d t ) = A t , (a t +c t ) = B t , (b t +d t ) = B t and a t +b t +c t +d t =N t . Equally, it is B t +B t = A t + A t = N t . In this context, it is p(a t )=p(A t B t ), p(A t ) = p(a t )+p(b t ) or p(A t )= p(A t B t )+ p(b t ) =p(A t B t )+p(A t B t ) while p(A t ) is not defined as p(a t ). In the same context, it is p(B t ) = p(a t )+p(c t ) = p(A t B t ) +p(c t ) and equally in the same respect p(B t ) = 1p(B t ) =p(b t )+p(d t ). Furthermore, the joint probability of A t and B t is denoted in general by p(A t B t ). Thus far, it is p(A t B t ) = p(A t ) -p(b t ) = p(B t ) -p(c t ) or in other words it follows clearly that p(B t ) + p(b t ) -p(c t ) = p(A t ). In general, it is p(a t )+p(c t )+p(b t )+p(d t ) = 1.

The data of the studies analysed
The data of the studies 10-12, 16-56 analysed, are presented by several tables (Table 2, Table 3, Table 5). The meaning of the abbreviations a t , b t , c t , d t , N t of tables ( Table 2, Table 3, Table 4, Table 5) is explained by a 2 by 2-table (Table 1). Some studies (Table 4) provided self-contradictory data and were not considered for a re-analysis.

EBV EBER-ISH and/or PCR Study of Hassani et al.
Several studies investigated whether EBV is present in the CNS with conflicting 57 results. Due to variations in terms of practical and technical approaches and other factors. Asma Hassani et al. 58 demonstrated ( Table 5) the presence of EBV in the involved tissues of the central nervous system of postmortem MS and non-MS autopsied human brain tissues.

Independence
In the case of independence of A t and B t it is generally valid that

Exclusion (A t Excludes B t and Vice Versa Relationship)
The mathematical formula of the exclusion relationship (A t excludes B t and vice versa) of a population was defined 15, 59-63 as (2) and used to proof the hypothesis: A t excludes B t and vice versa.

Necessary Condition (Conditio Sine Qua Non)
The mathematical formula of the necessary condition relationship (conditio sine qua non) of a population was defined 15, 59-63 as and used to proof the hypothesis:

Sufficient Condition (Conditio per Quam)
The mathematical formula of the sufficient condition relationship (conditio per quam) of a population was defined 15, 59-63 as (4) and used to proof the hypothesis: if A t then B t .

The X² Goodness of Fit Test of a Necessary Condition
Under conditions where the chi-square goodness of fit test 65 cannot be used it is possible to use an approximate and conservative (one sided) confidence interval known as the rule of three [65][66][67][68] . Using the continuity correction 69 , the chi-square value of a conditio sine qua non distribution before changes to (5)

The X² Goodness of Fit Test of the Exclusion Relationship
The chi square value with degree of freedom 2-1=1of the exclusion relationship with a continuity correction 69 can be calculated as

The Mathematical Formula of the Causal Relationship k
The mathematical formula of the causal relationship k 15, 59-63 is defined at every single event, at every single Bernoulli trial t, as where A t denotes the cause and B t denotes the effect. The chisquare distribution can be applied to determine the significance of causal relationship k. Pearson's concept of correlation 70 is not identical with causation, causation is not identical with correlation. In particular, the relationship between correlation and causation has already been discussed in many publications 71 . Thus far, repeating itself over and over again on this topic is only a waste of time and will not contribute anything new to further scientific progress.

The 95% Confidence Interval of the Causal Relationship k
A confidence interval (CI) of the causal relationship k calculated from the statistics of the observed data can help to estimate the true value of an unknown population parameter with a certain probability. The 95% interval for the causal relationship k is derived 63 as

The Chi Square Distribution
The following critical values 72 of the chi square distribution as visualized by Table 6 are used in this publication.

Null hypothesis
The presence of EBV VCA IgG antibodies is a necessary condition (a conditio sine qua non) of multiple sclerosis. In other words, the sample distribution agrees with the hypothetical (theoretical) distribution of a necessary condition.

Alternative hypothesis
The presence of EBV VCA IgG antibodies is not a necessary condition (a conditio sine qua non) of multiple sclerosis. In other words, the sample distribution does not agree with the hypothetical (theoretical) distribution of a necessary condition. The significance level (Alpha) below which the null hypothesis will be rejected is alpha=0,05.

Proof
The data reviewed by this article which investigated the relationship between EBV VCA IgG antibodies and multiple sclerosis are viewed by The presence of EBV EBNA1 IgG antibodies is a necessary condition (a conditio sine qua non) of Multiple sclerosis. In other words, the sample distribution agrees with the hypothetical (theoretical) distribution of a necessary condition.

Alternative hypothesis
The presence of EBV EBNA1 IgG antibodies is not a necessary condition (a conditio sine qua non) of Multiple sclerosis. In other words, the sample distribution does not agree with the hypothetical (theoretical) distribution of a necessary condition. The significance level (Alpha) below which the null hypothesis will be rejected is alpha=0,05.

Proof
The data reviewed by this article which investigated the relationship between EBV EBNA1 IgG antibodies and Multiple sclerosis are viewed by Table 3. Altogether, 32 studies with N=8003 cases and controls provided non selfcontradictory data and were considered for a meta-analyses while the level of significance was alpha = 0.

EBV is the cause of Multiple sclerosis
The presence of EBV DNA in the involved brain tissues is one appropriate way to show an etiological link between EBV and the pathogenesis of MS. Several studies performed published conflicting [73][74][75][76] results on this matter. Hassani et al. 58 carried out a study on autopsied human brain tissues and combined conventional PCR quantitative Taqman PCR targeting EBV BamH1 W fragment EBER-ISH and found that 91/101 (90%) of MS cases were EBV positive by PCR and/or EBER-ISH compared with 5/21 (24%) controls.

Claims Null hypothesis: (no causal relationship)
There is no significant causal relationship between an infection by Epstein-Barr virus and Multiple sclerosis. (k=0).

Alternative hypothesis: (causal relationship)
There is a significant causal relationship between an infection by Epstein-Barr virus and Multiple sclerosis. (k0).
The two tailed critical Chi square value (degrees of freedom = 1) for alpha level 5% is 3.841458821.

Proof
The data for this hypothesis test were provided by Hassani et al. 58 and are illustrated by the

DISCUSSION
Epstein, Achong and Barr 77 discovered 1964 a virus named Epstein-Barr virus (EBV). Soon after their discovery, Adams 78 and Nikoskelainen et al. 79 discussed already 1972 the possible relationship between EBV to Multiple sclerosis. The risk of acquiring this complex disease was linked to exposure to different environmental 80 factors among them non-infectious agents and infectious agents too. Thus far, several metaanalysis reviews [13][14]81 detected an association between EBV and MS through the investigation of antibodies, mainly EBV VCA IgG and EBV EBNA-1 IgG but without a definite solution.
This review is based on studies with as sample size of more than 8000 cases and controls. The retrospective nature of the studies may restrict our confidence to draw a generally valid and everlasting conclusion. Furthermore, another type of a limitation to consider is the definition used for classifying the viral status of a participant. Antibodies to various Epstein-Barr virus antigens were determined by different methods and individuals were considered EBV negative depending upon the preferences of the authors. In accordance with previous reports, is it possible at all to say anything generally valid under such circumstances?
For example, Gieß et al. 56 considered levels of EBV VCA IgG levels <20 U/ml as EBV VCA IgG negative and EBV VCA IgG levels > 20 U/ml as EBV VCA IgG positive with the consequence that 2 out of 100 MS cases were treated as EBV VCA IgG negative (false negative result).
Besides of the several different and severe limitations that must be acknowledged and which may contain several potential sources of bias the studies analysed agree on several points. All studies analysed support the hypothesis: without EBV VCA IgG or EBV EBNA1 IgG antibodies no Multiple sclerosis. Hassani et al. 58 studied autopsied human brain tissues and documented a necessary (Table 5) condition, a sufficient (Table 5) condition, a necessary and sufficient condition(