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This is the pre-peer reviewed version of the following article:-

Epstein-Barr and other viral mimicry of autoantigens, myelin and vitamin D related proteins, and of EIF2B, the cause of vanishing white matter disease: Massive mimicry of multiple sclerosis relevant proteins by the Synechococcus phage.

C.J.Carter

 which has been published in final form at Immunopharmacology and Immunotoxicology Posted online on April 12, 2011. (doi:10.3109/08923973.2011.572262)

 Abstract

The Epstein-Barr virus expresses proteins containing numerous short consensi (identical pentapeptides at least, or longer gapped consensi) that are identical to those in 16 multiple sclerosis autoantigens or in the products of multiple sclerosis susceptibility genes. Other viruses implicated in multiple sclerosis also display such mimicry and the Synechococcus phage was identified as a novel and major contributor to this phenomenon. Its cyanobacteria hosts favour temperate climes, in line with multiple sclerosis distribution, and bacterial and phage ecology accords closely with multiple sclerosis epidemiology. Bovine, ovine, or canine viral proteins were also identified as autoantigen homologues, in line with epidemiological data linking multiple sclerosis to cattle density, sheep contact and dog ownership.  Viral proteins align with known autoantigens, other myelin and vitamin D related proteins and the translation initiation factor EIF2B, which is implicated in vanishing white matter disease.  These data suggest that the autoantigens in multiple sclerosis, which causes demyelination in animal models, may be generated by antibodies raised to viral protein homologues. Multiple autoantibodies may cause multiple sclerosis via protein knockdown and immune activation. Their selective removal may be of clinical benefit as already suggested by promising results using plasmapheresis or immunoadsorption in certain multiple sclerosis patients.

Introduction

            Multiple sclerosis is a debilitating degenerative disorder characterised by peripheral and central nervous system demyelination, with consequent neurological defects. An autoimmune component is suggested by the presence of   autoantibodies targeting several myelin related proteins , including myelin basic protein (MBP), myelin associated glycoprotein (MAG), and myelin oligodendrocyte protein (MOG) or proteolipid protein (PLP), inter alia as well as cyclic nucleotide phosphodiesterase (CNP) and claudin 11 (1).  Certain features of multiple sclerosis can be induced in laboratory models of experimental autoimmune encephalomyelitis by inoculation with multiple sclerosis autoantigens, including MBP, PLP, MAG, and MOG, suggesting a key role for autoantibodies in the genesis of demyelination (2). 

A viral cause of multiple sclerosis has often been suspected, most commonly, the Epstein-Barr virus, (3) . Several viruses including adenoviruses, herpes simplex, coxsackie viruses and the common cold rhinoviruses have also been associated with relapse in this condition (see supplementary file msrisk.htm). Viral and bacterial mimicry  of the autoantigens in multiple sclerosis has also been reported suggesting that pathogen antibodies could also target the myelin-related and other autoantigens in the disease (4-6) .

            The large scale integration of non-retroviral DNA into mammalian genomes has recently been demonstrated (7) . Both DNA and RNA viruses have contributed to this phenomenon, the latter via the prior conversion of RNA to DNA by genomic retrotransposons (8) . Viral RNA genomes have also been incorporated into those of plants, arthropods, fungi, nematodes, and protozoa (9) . Convergent viral evolution may also be in part responsible for these viral/human DNA matches.

As a consequence of such integration or mimicry, current viral proteins resemble our own. This phenomenon has been noted for hexapeptide matches for the influenza virus (10)  and pentapeptide or greater human matches for 30 other common viruses infecting humans (11;12). Bacterial proteins show even greater homology to human proteins and the coverage of the human proteome by both viral and bacterial consensi is total (13;14). In other words, every single human protein is homologous to a spectrum of viral or bacterial proteins. Bioinformatics analyses posted at http://www.polygenicpathways.co.uk/blasts.htm show that each virus (of 28 examined) expresses viral matches (vatches) homologous to a large but specific spectrum of human proteins and that each human protein examined expresses vatches to a large but specific spectrum of viruses.   The herpes simplex virus, for example, expresses proteins containing consensus peptide stretches that are highly homologous to the products of multiple Alzheimer’s disease susceptibility genes (13).  

Viral proteins bind to hundreds of host proteins during their life cycle, as recorded for herpes simplex at http://www.polygenicpathways.co.uk/herpeshost.html and in the VirusMint interaction database (15), an ability no doubt related to this homology that allows them to compete with their human counterparts as protein interactors. In so binding, viruses are able to interfere with the signalling networks and interactomes of a variety of key human proteins.  A recent analysis of the host/pathogen interactions of 110 viruses showed nodes of interactions where the host proteins involved are clearly related to diverse human diseases (16).

Viral vatches can be identified by comparing human proteins with viral proteomes, while human vatches can be identified by comparing translated viral genomes with the human proteome. This exercise, using multiple sclerosis autoantigens, or the Epstein-Barr virus, as baits reveals multiple suspected and novel viral culprits and many novel potential autoantigens relevant to multiple sclerosis.

Methods.

            The protein sequences of certain autoantigens implicated in multiple sclerosis (cyclic nucleotide phosphodiesterase (CNP), Claudin 11 , myelin associated glycoprotein (MAG), myelin oligodendrocyte protein (MOG) and myelin basic protein (MBP) identified from a recent antigen microarray study in Multiple sclerosis (1) ) were compared with those of all viral proteomes using the BLAST server at NCBI (blast p) (17).

 A more detailed analysis, identifying and counting the numbers of vatches in MBP, MOG and claudin 11 (aka oligodendrocyte specific protein) was undertaken, and the results are available in supplementary file msants.xls. From this analysis, the Synechococcus phage emerged as the primary vatch contributor and this trail was followed by BLAST analyses of the translated Synechococcus phage genome.  The Epstein-Barr or Synechococcus phage translated genome was compared to the human proteome with or without the Entrez filter “multiple sclerosis” or others (nucleotide vs. protein, BlastX) and the EIF2B5 translation initiation factors subunit compared against all viral proteins. B-Cell and T cell epitopes were identified using the Immune epitope database server http://tools.immuneepitope.org/main/index.htm which predicts the antigenicity of peptide sequences, based on their charge and hydrophobicity properties (18;19) . Because of the large number of proteins, unless referenced, the brief notes describing the role of the protein are culled from the summary section of Entrez gene.  References relating to the role of viruses in multiple sclerosis, together with a list of reported autoantigens, are stocked in supplementary file msrisk.htm. The BLAST results of this exercise are posted at http://www.polygenicpathways.co.uk/msblasts where the analyses can be verified by the reader. A BLAST result of the Synechococcus phage that illustrates the vatches in myelin-related proteins is provided at http://www.polygenicpathways.co.uk/synechomyelin.htm. Multiple sclerosis susceptibility genes (any with at least one reported positive association) were identified from MsGene http://www.msgene.org (20) and from genome-wide association data at http://www.genome.gov/gwastudies/ (21) .

Statistics

The BLAST algorithm is designed to detect overall homologies between whole viral and human proteins, rather than intra-protein identities and pentapeptide or similar identities within proteins are not considered as significant by the server. In isolation, such perfect matches are evidently significant. The significance of enrichment within datasets was analysed by the Chi squared test, assuming expected values of: genes in genome = 35,000, multiple sclerosis susceptibility candidates = 328 and number of autoantigens = 37.  The figure of 35,000 approximates to the estimated number of human proteins (22) , and the figure of 328 susceptibility genes is from MsGene. The 37 autoantigens are referenced in Supplementary file msrisk.htm. In any dataset of genes one would expect 328/35000 to be multiple sclerosis susceptibility genes and 37/35000 to be autoantigens, providing the expected values for the chi squared test.

Results

Viral homologues of the autoantigens cyclic nucleotide phosphodiesterase (CNP), Myelin associated glycoprotein (MAG), myelin basic protein (MBP) myelin oligodendrocyte protein (MOG) and Claudin 11 (also known as  oligodendrocyte specific protein) include the Epstein-Barr virus and a host of others implicated in relapse (Coxsackie virus, herpes simplex, influenza , herpes virus 6,  inter alia) or which have been detected in multiple sclerosis sera or CSF, or which are known to cause demyelination (Fig 1, Table 1 and supplementary file msants.xls). A number of bovine, ovine, canine, feline and other agricultural viral proteins are also homologous to these proteins, in line with certain epidemiological studies linking animal contact or distribution to multiple sclerosis (see below). Many phage or plant viruses, whose contribution to multiple sclerosis has not been considered, also express autoantigen vatches. Although many of these are unlikely to infect humans, all are included to show the extent and variety of this phenomenon.

Conversely, the Epstein-Barr virus, or the Synechococcus phage (see below), express proteins which are homologous to the autoantigens, as well as to the products of many multiple sclerosis susceptibility genes (Fig 2). These targets are highly and significantly enriched within these datasets (using the BLAST filter “multiple sclerosis”) (p < 0.0026 for the autoantigens (but see also Table 2) and P < 1e-30 for the genes).  One of the targets of the Epstein-Barr virus was the translation initiation factor EIF2B4. The targeting of the EIF2B subunit is of particular interest as mutations in EIF2B5 and EIF2B2 are responsible for vanishing white matter disease, which leads to intense cerebral demyelination (23) . Because of this, the EIF2B5 subunit was screened against all viral proteomes (Fig 1). A number of phages and viruses contain EIF2B5 vatches. Those predicted to be highly antigenic include proteins from the Coxsackie virus and Staphylococcus phage, streptococcus and Bacillus phages, polyomaviruses, Hepatitis delta; Herpes virus HSV-7, the human metapneumovirus and human and canine noroviruses (see supplementary data msvatches.htm). Listeria infection is known to cause demyelination in chickens (24) , and  both adenoviruses and hepatitis C have been linked to multiple sclerosis relapse, found in serum or CSF (25;26)   or provoke disease resembling multiple sclerosis (Hepatitis C) (27) .

            The total number of vatches per virus in MBP, MOG and claudin 11 were counted from the supplementary table msants.xls which also correlates the viral/human alignments with the B or T cell antigenicity of the autoantigen or its matching partner.  The diverse and extensive panoply of human, animal, fish, invertebrate, plant and bacterial viruses (phages) and other exotica that mimic vatches in MBP, MOG and claudin 11 autoantigens is shown in Table 1. Again, many of these have been implicated in multiple sclerosis, either as risk factors, associated with relapse, or detected via antibodies or DNA methodology. Others have been associated with demyelinating diseases. A number of bovine, ovine, canine and feline viruses figure among these lists as do lactococcus and lactobacillus phages. Multiple sclerosis risk has been associated with unpasteurised milk, bovine geographical density, contact with sheep, and dog and small pet ownership (28-30) The presence of a number of poultry, equine and porcine viruses within these lists suggests that other zoonotic viruses could well contribute to the aetiology of multiple sclerosis or demyelination.

While plant and other related viruses are not generally believed to cause disease, a pepper mottle virus is known to cause fever and an immune response in Man (31) and such diseases may well be more common than thought, particularly in the light of this type of homology. These viruses, as well as those in foodstuffs (e.g. meat, fish, shrimp and oysters) can evidently be ingested, inhaled and absorbed through cuts, while our extensive bacterial microbiome is host to a vast number of bacteriophages.

By far the most prevalent vatches, in any class, occurred in two phages infecting Synechococcus and Prochlorococcus cyanobacteria (Table 1). These are discussed at length below.

Because of this, both the Epstein-Barr virus and the Synechococcus phage translated genomes were screened against the human proteome using various filters. Although other common viruses express as many, or more matching proteins (Table 1) the Epstein-Barr virus was chosen because of  repeated studies implicating this virus as a risk factor in multiple sclerosis (see introduction).  Both viruses express vatches (often predicted to be immunogenic) to a large number of myelin related proteins, to 16 autoantigens, to all subunits of the EIF2B translation initiation complex and to vitamin D related proteins (Table 2).  Reduced levels of vitamin D, and low sunlight exposure, which plays a role in vitamin D synthesis, have been implicated as risk factors in multiple sclerosis (32) .The myelin targeted BLAST for the Synechococcus phage is available at  http://www.polygenicpathways.co.uk/synechomyelin.htm and annotated further results in supplementary file synechotable.htm

Unfiltered BLAST results

The homologies above were trawled using filters (multiple sclerosis, myelin, etc.) that orient the results towards the requested targets. The results returned from unfiltered BLASTs return the most homologous proteins and provide an idea of the viral targets of choice.

The Epstein Barr virus (Table 3) encodes for a number of proteins that are similar to DNA polymerase subunits and other enzymes that are no doubt relevant to its implication in cancer (33), which are not discussed further. It also encodes for an interleukin 10 homologue, which is relevant to multiple sclerosis by virtue of its key role in the immune network. Human IL10, a key multiple sclerosis susceptibility gene, is both anti-inflammatory and immunosuppressive (34). The role of this highly homologous IL10 viral protein does not appear to have been studied in relation to multiple sclerosis although infection with such a close mimic must surely impact upon the immune network. Apart from this, the unfiltered Epstein-Barr viral BLAST did not preferentially return either multiple sclerosis genes, apart from IGH@, or autoantigens (Fig 2) (non-significant enrichment). However the involvement of the virus in cancer and several autoimmune disorders  (systemic lupus erythematosus , rheumatoid arthritis, and Sjogren's syndrome) (35) as well as atherosclerosis (36), inter alia,  suggest that its predilection may lie elsewhere, despite a close link with multiple sclerosis. It should also be noted that these BLASTs are limited by the NCBI server to 20,000 alignments, a figure that was always exceeded, and that filtered BLASTS (e.g. using the term multiple sclerosis) return a further set of human proteins that contain multiple viral/human consensi (see Table 1).

Similarly, the Synechococcus phage possesses proteins that might also be involved in cancer (replication factors) (Table 4). The potential contribution of this phage to such conditions has not been studied, and indeed the role of bacteriophages in human diseases appears to have been little studied in general.  The Synechococcus phage also encodes for a number of enzymes that are highly homologous to glucose and hexose-6-phosphate dehydrogenase (a multiple sclerosis susceptibility gene) and phosphogluconate dehydrogenase. G6PD is important for the maintenance of the levels of the antioxidant, glutathione (37). Glutathione depletion has recently been observed in the multiple sclerosis brain (38). Oligodendrocytes are particularly sensitive to glutathione depletion (39), and the cytotoxic effects of various insults can be reversed by addition of the glutathione precursor N-acetyl cysteine (40).  Again the presence of such a viral mimic is likely to impact upon the function of the human enzyme and its metabolic pathways.

The unfiltered Synechococcus phage BLAST also targeted several (8) multiple sclerosis susceptibility genes (3.7 fold enrichment P < 0.0001), and 6 autoantigens (24 fold enrichment P < 1e-30) including MAG, MBP, and MOG. Antibodies raised to these proteins in the experimental autoimmune encephalomyelitis model, cause demyelination (2). Thus, although other proteins are also targeted, the Synechococcus phage shows a clear predilection for multiple sclerosis related proteins.

While it seems unlikely that phage DNA (or plant DNA) would be inserted into the human genome, the viral and human proteins remain highly homologous. Such events also have to be reasoned in relation to evolutionary time, when our ancestors were simpler multicellular organisms. Viruses are also inter-related and such homology could stem from this, and from convergent viral evolution.  

Protective phages and viruses?

Hepatitis B and cytomegalovirus infections may protect against multiple sclerosis while Lactobacillus infection has beneficial effects in animal models of multiple sclerosis (41-43) although the consumption of unpasteurised milk has also been linked to the distribution multiple sclerosis (30). Hepatitis B, the cytomegalovirus and various lactobacillus phage proteins align with multiple sclerosis autoantigens. There may therefore be some subdivision of these phages and viruses into good or bad. Beneficial viruses may perhaps exert their effects by blocking T-cell activation, acting as antagonists to their human counterparts, while toxic vatches may exert their effects via immune-related protein knockdown or autoimmune activation and cellular destruction.  

The risks of vaccination

Hepatitis B vaccination has been reported (rarely) to induce  a condition resembling multiple sclerosis (42) and the virus expresses vatches homologous to EIF2B5, while multiple vaccinations against the papillomavirus virus , which expresses CNP homologous vatches, have also been implicated in demyelination (44).  Such effects of vaccination could perhaps be reduced by ensuring that future vaccines do not contain these matching epitopes.

Synechococcus and Prochlorococcus phages.

These phages are double stranded circular DNA phages infecting Synechococcus and Prochlorococcus cyanobacteria which encompass several related species of marine and freshwater bacteria with a worldwide distribution. They are the most abundant members of picophytoplankton in the marine environment. Prochlorococcus is more abundant in warm (> 15°C) waters, while Synechococcus species are predominant in coastal and more temperate open ocean waters. Synechococci are found in well-lit surface waters in the ocean, and close to the shore, and are prolific at higher water temperatures near the surface (45).

Anacystis nidulans is a freshwater form of Synechococcus. A.Nidulans thrives on nitrates, phosphates, sulphates and urate and is one freshwater species responsible for harmful algal blooms (46-49). Nitrate water levels have been linked to the distribution of multiple sclerosis (50). Its growth is inhibited by copper, cadmium and silver (51;52) and dependent on iron (53). The distribution of multiple sclerosis has been reported to be related to the presence of soluble iron in the soil in Finland (54). While heavy metals are toxic to the bacteria, (causing phage release); they have a different effect on their phages. For example the growth of an A-1 cyanophage in A.Nidulans is stimulated by copper, causing lysis of its bacterial host and phage release (51). Clusters of multiple sclerosis have occasionally been reported close to areas of high heavy metal contamination in water and sewage plants (55) and the distribution of multiple sclerosis has also been linked to relatively high water concentrations of  barium, calcium, chloride, chromium, magnesium, manganese, molybdenum,  strontium and zinc (50).

            Ultraviolet light can stimulate the growth of A.Nidulans (56), but prolonged exposure reduces its viability , an effect that can be reversed by a shift to red light conditions (57).  Phages are also released from A.Nidulans by UV radiation (58) . Ultraviolet light is concentrated at the equator while red light predominates at sunset and sunrise and when the sun is low on the horizon, as at higher latitudes away from the equator. Together, these effects correlate closely with the geographical distribution of multiple sclerosis which is more prevalent at higher latitudes and in areas of low ultraviolet radiation (59) . These effects are also related to the necessity for sunlight to stimulate Vitamin D production. Lower levels of this vitamin, again related to a latitudinal gradient,  have also been implicated in multiple sclerosis risk (60), as have vitamin D related gene targets (61).Vitamin D also plays a role in the immune system and stimulates the production of bactericidal proteins (62). Again this highlights relationships between genes environmental factors and the immune system which may condition each other’s effects. It has also been noted that the distribution of multiple sclerosis correlates well with the degree of exposure to near horizon sunlight (63).

            Although the distribution of multiple sclerosis is skewed towards temperate climes, eruption of disease activity is closely correlated with higher temperatures from March to August in the USA (64). This effect has also been observed in Italy where, in the summer months, relapse hospital admissions correlate synchronously with high temperature and low wind speed (65). High temperatures stimulate the growth of these cyanobacteria, while UV light favours phage release. High temperature and low wind speed are favourable conditions for visits to rivers, pools, lakes and beaches.

            A recent study in Bosnia and Herzegovina has shown that multiple sclerosis prevails in inland municipal areas being almost absent in coastal regions (66). Lower coastal incidences of multiple sclerosis have also been reported in Ecuador, Croatia, France and Norway(67-70).  Although counter to the above, as residents in coastal regions would be exposed to the marine environment at an early age, this could suggest that immunisation might be a consequence of childhood exposure to these agents. If such is the case, repeated exposure to these agents at an early age may lead to immune tolerance and recognition of the viral antigens as self, a possibility that suggests a route for vaccine development.

            While this evidence is circumstantial, the several diverse converging lines of evidence and the extensive homology of the Synechococcus phage to myelin related proteins and autoantigens suggests that this and related phages could be major contributors to the aetiology of multiple sclerosis.

Multiple sclerosis genes and the Epstein-Barr virus and Synechococcus phage.

A large number of multiple sclerosis susceptibility genes encode for products homologous to Epstein-Barr and the Synechococcus phage and both datasets (with the filter multiple sclerosis) are heavily enriched in multiple sclerosis genes (Over 20 fold: P < 1e-30: Fig 2). The unfiltered BLAST also showed that the Synechococcus phage dataset is highly and significantly enriched in multiple sclerosis related gene products (P < 0.0001) and autoantigens (P< 1e-30).

This  marriage of genes to risk factors suggests that the genes may be risk factors precisely because they encode for products homologous to those of the viral risk factors, and vice versa. This could perhaps be addressed by partitioning GWAS data in relation to the predicted sequences of autoantigens present in patients.  

            The human proteome has been estimated to contain ~33,869 proteins with an average length of 375 amino acids (22) (not counting the splice variants). For pentapeptide matches, this yields a figure of 370*33869 potential matching blocks (12.53 million). These building blocks are identical to those in viral proteins, as described above, and also to bacterial, fungal and parasite proteins.

A recent study of bacterial/human matching from 40 pathogenic and non-pathogenic bacteria demonstrated 47,610 total identical nonapeptide bacterial protein matches found in 10,701 human proteins (71). In a further study, 5,260,383 perfect pentapeptide bacterial protein matches were distributed through 36,014 human proteins (14); In other words, every single human protein contains multiple peptide matches to diverse species of bacteria. The same is true with respect to viral proteins, and each human protein contains consensus sequences from a large but specific spectrum of viruses, while viral proteins contain consensus sequences to a spectrum of human proteins (13) .

With the power of modern day bioinformatics, it should soon be possible to match human and pathogen convergence to disease.

On the absence of viral seropositivity or DNA.

As can be seen from the B-call and T-cell antigenicity analyses of the autoantigens the supplementary table msants.xls a single human protein can contain several epitopes, and the same is true for viral proteins. The Epstein-Barr virus expresses 80 proteins, which may be differentially expressed during various phases of the life cycle.

In the clinical setting, viral detection is usually performed using a single antibody that is capable of detecting the virus, if present.  If antibodies raised to the virus have successfully eliminated the pathogen, evidently, immunochemical or DNA detection methods will be negative. Successful viral elimination may have come at the cost of collateral damage produced by autoimmune and inflammatory activation. If certain anti-viral antibodies also target human proteins, their production will be maintained due to the continual presence of the human homologous autoantigen. Indeed, because of this viral/human homology, one could argue that the autoantibodies themselves are evidence for previous, but currently extinct viral infection. It is known that antibodies to the Epstein-Barr virus cross react with MOG and other myelin related autoantigens (72;73).

The past or current presence of the virus could only be adequately assessed using a large panel of antibodies to different epitopes and by testing each for viral/human cross reactivity. Because of such limitations, the absence of a virus, using antibody or DNA methodology, need not preclude its earlier presence.

Multiple sclerosis related genes and viral life cycles

Many of the genes associated with schizophrenia or Alzheimer’s disease are also involved in the life cycles of the pathogens implicated as risk factors, and the pathogen’s human  binding partners impinge upon signalling networks relevant to the disease (74;75) .  A survey of the Epstein-Barr viral life cycle shows that this is also the case in multiple sclerosis. Genes implicated in multiple sclerosis are highlighted in bold in this section (from MsGene). For example the Epstein Barr gp42 protein binds to the top MsGene susceptibility gene product HLA-DRB1 (76) , while the viral protein EBNA-3 binds to the vitamin D receptor, VDR (77) . The Complement receptor 2 (CD21 or CR2) is also a viral receptor (78) relevant to various complement components implicated in multiple sclerosis (C4A, C5, C7).

 The virus also expresses a protein that is highly homologous to IL10, while the viral latent membrane protein encodes for a mimic of CD40 (79). The viral latent membrane protein 1 (LMP1) also functions as a constitutively activated receptor of the tumour necrosis factor (TNF) receptor family (80) and activates the signal transducer and activator of transcription  STAT3  (81). LMP1 also binds to tyrosine kinase TYK2 and inhibits interferon signalling (82).  Interferon alpha’s (IFNA2, IFNA17) or gamma (IFNG) , interferon receptors (IFNAR1,IFNAR2 , IFNGR1 ),  interferon regulated proteins (IFIH1, IFIT1) or regulatory factors (IRF1, IRF5, IRF8) have all been associated with multiple sclerosis and viral LMP1 expression is controlled by the interferon regulatory factor IRF5 (83).  The viral protein BHRF1 inhibits apoptosis by binding to a number of pro-apoptotic proteins (BID, PUMA and  BAK) all of which interact with the apoptosis regulatory protein BCL2  (84), while the viral protein BZLF1 inhibits the expression of the TNF receptor TNFRSF1A (85) . The Epstein-Barr virus is reactivated by the transforming growth factor TGFB1 (86). Protein kinase R (EIF2AK2) is a double stranded DNA viral detector involved in the reaction to many viruses (87). The APOE genotype also determines susceptibility to infectious mononucleosis caused by the Epstein-Barr virus (88). The Synechococcus phage also expresses a protein that is highly homologous to H6PD

This incomplete survey illustrates some of the multiple sclerosis susceptibility genes that are influenced by or influence the Epstein-Barr virus, and such interactions are likely to condition the risk-promoting effects of both genes and virus.

 Discussion

The natural world abounds with hundreds of viruses expressing vatches identical to those in multiple sclerosis autoantigens. It is evident from this and other analyses that single human proteins contain vatches identical to those in dozens of viruses and bacteria and that single viruses and bacteria express vatches identical to those in hundreds of human proteins. The viral and bacterial coverage of the human proteome is total and no protein is exempt (12-14).  These matching sequences can be identical, for example pentapeptides or more, as observed by other authors (12) or longer gapped consensi (see Table 2). From an immunological perspective, one could argue that the slightly differing consensi are dissimilar enough to be regarded as non-self, but similar enough for their antibodies to also target the human homologue, and thus the more malignant.  Epitope mapping in relation to human/viral cross-reactivity would be useful in this respect. Although multiple viral/human vatches exist, the overall spectrum of the viral to human and human to viral vatches is distinct (see Figs 1 and 2 and the supplementary table msants.xls Viruses are implicated in many diseases, and these spectra, and the function of their human targets may define the diseases likely to be provoked by viral interference.   In multiple sclerosis, these viruses include those already implicated as risk factors and a host of others whose risk promoting or protective effects can only be guessed at. This survey clearly implicates the Epstein-Barr virus in multiple sclerosis and confirms a role for many others that have been reported to affect onset or relapse (e.g. Influenza, herpes simplex, Coxsackie viruses and rhinoviruses, inter alia).

The survey also identified a number of canine, feline, bovine and ovine viruses expressing vatches common to multiple sclerosis autoantigens. Such homology may well explain the reported relationships of multiple sclerosis to dog and small pet ownership as well as to sheep contact and bovine density. The possibility of other zoonoses (equine, porcine or poultry viruses, or even bat contact) cannot be ruled out, nor can the presence of viruses in our diet, given the homology of their proteins to multiple sclerosis autoantigens. While plant viruses are generally considered as innocuous, the example of the Synechococcus phage illustrates the realms of the possible.

Although the Epstein-Barr virus extensively mimics multiple sclerosis proteins, supporting a role for the virus as a risk factor or causative agent, many other viruses share this property. This diversity likely explains many of the controversies in relation to viral risk factors. Where the Epstein-Barr virus is not involved, there are a host of other phages or viruses, to take its place. Antibodies to bacterial peptides have also been shown to cross-react with MBP and MOG (89).

In addition, antibodies to the virus may persist, due to sustained encounters with the human homologue autoantigens, even after elimination of the virus.  These mimics target various autoantigens from the same known panel. This type of vatch mining is thus able to confirm the role of current viral suspects and to identify many others that may have a key role in the pathology of multiple sclerosis. This approach is also able to predict other potential autoantigens that are highly relevant to multiple sclerosis, for example the myelin or oligodendrocyte transcription factors, vitamin D related proteins, and the translation initiation factor subunits of EIF2B. BLAST analysis of the EIF2B5 subunit in turn identified novel viral suspects, particularly the Listeria phage and adenovirus 50.

Vatches may exert their effects via immune related knockdown of their human counterparts, and may also act as mimics, for example IL10 , dummy ligands, or decoy receptors or enzymes , for example G6PD, and may also interfere with the target protein’s interaction network via theft of its usual binding partners They may also trigger an autoimmune response to their human homologues causing cellular degeneration via immune and inflammatory attack (13;90) and this may well be the case in multiple sclerosis. It is already accepted that multiple sclerosis is an autoimmune disorder, but this survey may help to identify the most important viruses and autoantigens. These might perhaps be targeted, eventually by vaccines, and perhaps by anti-antibody antibodies or by immunisation with epitopes from the apparently beneficial viruses.        

This analysis infers that multiple sclerosis may be caused by pathogen-derived autoantibodies, whose formation is both genetically and immunologically conditioned.  The concept of antibody toxicity has already been largely proven by the models of experimental allergic encephalomyelitis induced by antibodies to myelin antigens, (PLP, MAG and MOG inter alia) (2) initially developed by inoculation of rhesus monkeys with brain extracts in 1933 (91). While these models may not exactly mimic the human condition, this may relate to the fact that several autoantigens can be observed in multiple sclerosis, while the laboratory models tend to focus on one.

Specificity and conditional effects.

Many are infected with the Epstein-Barr virus, or with other common viruses affecting multiple sclerosis or relapse, but do not develop multiple sclerosis. A host of viral proteins from human, bacterial, plant and animal viruses not (yet?) implicated in multiple sclerosis also express proteins that are homologous to myelin related proteins.

How can this diversity be reconciled to an autoimmunity theory, positing that viral homologues create the autoantibodies in multiple sclerosis? Although this is backed up by the homologies of the Epstein-Barr virus, and many others implicated in multiple sclerosis, to key myelin-related proteins, many other viruses share this property.  In the case of the Synechococcus phage, the ecology of it and its host correlate well with the known epidemiology of multiple sclerosis, and perhaps other examples exist within this extensive panoply. The presence of bovine, ovine, canine and feline viruses within this spectrum may also explain the links of multiple sclerosis to bovine density, contact with sheep, and dog and small pet ownership.

Viruses will not always raise antibodies that also target their human homologues and clues to this conundrum perhaps lie in human susceptibility genes that determine protein sequences, and therefore the extent of homology to viral proteins, and in the large number of  immune-related genes that have been associated with multiple sclerosis (92;93). The concentration of multiple sclerosis related genes in the viral datasets hints at such conditioning.

In Sjogren’s syndrome, different HLA alleles are associated with the generation of autoantibodies to the SSA and SSB autoantigens (94). The propensity for developing autoantibodies to particular proteins is also genetically determined and inherited (95).  The recognition of self and non-self is also determined and imprinted soon after birth. Maternally transferred antibodies via the placenta, or after birth via colostrum may also influence the subsequent development of the immune system (96).

Risk factor and genetic association studies are notoriously inconsistent, but the evident marriage of genes and risk factors suggests a model that embraces such heterogeneity. 

The production of antibodies deleterious to the function of protein X will depend upon a number of factors including our genes, that determine protein sequences, whether, when and which strain of pathogen is encountered, whether our genes aid or prevent infection by interfering with pathogen life cycles, the extent of the epitope match between human and pathogen protein, whether or not this match is deemed to be self or foreign, how many  infections from diverse agents fulfil these criteria, immune-related genes, and whether or not the ability to raise autoantibodies has been inherited.  Many of these factors are interdependent and, rather like a casino slot machine, the final extent of risk may be determined by the number of matching reels. Because so many factors may be conditional upon each other, heterogeneity when testing single risk factors in isolation is to be expected. It may be possible to partition GWAS data in relation to some of these variables, to test such multiple interactions.

Immunopharmacology: Antibody-mediated knockdown as a pathological mechanism.

The symptoms of multiple sclerosis can be reliably produced in mice by various transgenic knockouts; for example MAG, MBP or myelin P0 knockouts impair myelination (97;98). While the expression of such proteins may be reduced in multiple sclerosis, due largely to white matter loss (99), there is little to suggest total or partial overall protein knockout as seen in the transgenic models. However, functional protein knockdown could well be accomplished by antibodies to these proteins, a situation rendered possible by the ability of antibodies to traverse the blood-brain barrier (100) and also to enter cells (101).  Given the viral homology to so many autoantigens , it thus seems likely that viral antibodies, that also target human proteins are the causative agents in multiple sclerosis, producing their effects via antibody-mediated knockdown of multiple proteins relevant to the disease process, a scenario that is independent of immune-related processes, where the antibodies are acting simply as antagonists.  Such an effect would prevail even during immunosuppression, unless the antibodies were eliminated, a factor that perhaps explains the relative lack of success of immunosuppressive strategies in multiple sclerosis (102). 

Autoantibody removal as a therapeutic strategy

Coastal residence appears to reduce the incidence of multiple sclerosis in some cases, a factor that could tentatively be related to the development of immunity to marine Synechococcus or Prochlorococcus phages, presumably via a route that does not involve autoantibody generation. Studies of this phenomenon could perhaps lead to a vaccine for multiple sclerosis. Desensitisation procedures might also be considered in the research setting. These data also suggest that autoantibodies are a cause, and not a consequence of multiple sclerosis. If such is the case, then removal of these antibodies by dialysis over an affinity column laced with appropriate antigens and antibodies, tailored to each individual’s autoantigen profile, might be expected to stem the progression of multiple sclerosis, and to favour the success of other curative regimes such as stem cell therapy. In fact plasmapheresis, which exchanges plasma (containing antibodies) with synthetic or donor plasma, was initially shown to be of clinical benefit in a small (16 patients) trial over 20 years ago in Japan. Muscle power improved in all patients and a proportion also showed amelioration in sitting, standing and walking ability   while most reported benefits in standing still, sensory disturbance, visual disturbance, and urinary complications (103). However, a recent retrospective analysis reported that such treatment was ineffective in multiple sclerosis, but beneficial in steroid-resistant exacerbations in relapsing forms of multiple sclerosis (104).  Plasma immunoadsorption which removes immunoglobulins, immune complexes, and complement from plasma has also been reported  to delay disease progression and to ameliorate symptoms in multiple sclerosis in a number of studies (105-109).

Plasmapheresis removes all antibodies, and a number of other plasma–borne proteins, which may be either malevolent (e.g. autoantibodies and pro-inflammatory cytokines) or beneficial (e.g.  Growth factors or protective antibodies or cytokines), while immunoadsorption may also remove beneficial antibodies, or immune complexes. The results of both plasmapheresis and immunoadsorption are promising but a more targeted approach aimed specifically at autoantibodies, perhaps tailored to each individual’s autoantigen profile might well be more effective.

Further research on these homologous pathogen/human proteins, and the development of antibody array technology on the same scale as genome-wide association studies would greatly aid out understanding of the role of viruses and other pathogens, the microbiome and autoimmunity in multiple sclerosis and many other diseases.

 

 

 

Table 1:  The overall number of viral vatches (N in brackets) per species for combined data from the MBP, MOG and claudin 11 autoantigens the supplementary table msants.xls Those in bold text  have been implicated in multiple sclerosis (associated with the disease or with relapse, or found in multiple sclerosis (antibodies or DNA) or cause demyelination. Phage bacterial hosts that have been found in multiple sclerosis sera or CSF or animal species (cattle, sheep, dogs and household pets) that have been implicated as risk factors are also highlighted in bold (see supplementary file msrisk.htm.)

 

Human	Animal	Plant, bacteria  and others
HIV-1 (15) Influenza A (13) Enterobacteria phage (12) Hepatitis B (10) Bacillus phage (9) Cytomegalovirus(9) Hepatitis C (8) Poliovirus (8) Human rhinovirus (common cold)(8) Mycobacterium phage (7) Staphylococcus phage (6) Dengue virus (6) Klebsiella phage (5) Measles (5) Human Coronavirus (5) Enterovirus (5) Acinetobacter phage (5) HSV-2 (4) Saffold virus (4) Norovirus (4) Lactococcus phage (Milk) (4) Clostridium phage (4) Epstein-Barr virus (4) Saffold virus (4) Salmonella phage (4) Human adenovirus 4 (4) Pseudomonas phage (3) Rotavirus (3) JC Polyomavirus (3) Adeno-associated virus (3) Norwalk virus (3) Human astrovirus (3) Hepatitis delta (3) Human herpesvirus  8 (3) Vibrio phage (3) Enterococcus phage (3) Burkholderia phage (3) Listeria phage (2) Zika virus (2) Torque teno virus (2) GB virus (2) Shigella phage (2) Wesselsbron virus (2) Human coxsackievirus (2) Streptococcus pyogenes phage (2) BK polyomavirus (2) Human parvovirus (2) Varicella (2) Chapare virus (2) Serra do Navio virus (2) West Nile virus (2) St. Louis encephalitis virus (2) Lacrosse encephalitis (2) Tensaw virus (2) HHV-7 (2) Rubella (2) HHV-7 (2) Yersinia phage (2) Toscana virus (1 and all below) HMO Astrovirus Torque teno mini virus Vaccinia virus Variola virus Polyomavirus HPyV7 Human herpesvirus 6 Influenza C Fiji disease virus Bunyamwera virus Chiba virus Moussa virus Tanapox virus Torque teno sus virus	Bat coronavirus (8) Infectious bronchitis virus (Poultry)(7) Bovine viral diarrhea virus (7) Meleagrid herpesvirus 1 (poultry) (6) Feline coronavirus (6)  Grouper iridovirus (Fish) (5) Oliveros virus (Rodents) (5) Orange-spotted grouper iridovirus (Fish) (4) Lymphocystis disease virus (Fish) (4) Alcelaphine herpesvirus (cattle) (4) Gallid herpesvirus 1 and 2 (Poultry) (4) Shrimp white spot syndrome virus (3) Porcine reproductive and respiratory syndrome virus (3) Equid herpesvirus 9  Ovine herpesvirus 2 (3) Fowlpox (3) Pipistrellus bat coronavirus (3) Canine papillomavirus (3) Swine fever virus   Theiler's encephalomyelitis virus (3) Visna Maedi virus(sheep) Deerpox virus (2) Glossina pallidipes salivary gland hypertrophy virus (2) Turkey coronavirus  (2) Marseillevirus (2) Fowl adenovirus 4 (2) Peste-des-petits-ruminants virus (sheep) (2) Fowl adenovirus 4   (2) Foot and mouth virus (cattle) (2) Feline herpesvirus 1  (2) Turbot reddish body iridovirus (2) Equine papillomavirus (2) Psittacid herpesvirus 1 (Parrots) (2) Tamana bat virus (2) Equine coronavirus (2) Ostreococcus tauri virus (Oyster) (2) Ostreid herpesvirus 1 (Oyster) (2) Orange-spotted grouper iridovirus (Fish)(2) Bluetongue virus (cattle)(1) Bovine herpesvirus 4 Sandfly Sicilian Turkey virus Wood mouse herpesvirus Apodemus flavicollis rhadinovirus (mouse) Avian paramyxovirus Ornithogalum mosaic virus Adoxophyes honmai NPV Ateline herpesvirus 3 (monkey) Porcine epidemic diarrhea virus Drosophila affinis sigma virus Infectious hematopoietic necrosis virus (Fish) Avian leukosis virus Bovine herpesvirus 5 Cowpox Rabbitpox Ectromelia virus Porcine teschovirus Swinepox Infectious salmon anemia virus Ovine enzootic nasal tumor virus Small ruminant lentivirus (sheep) Bluetongue virus (cattle) (1) Porcine lymphotropic herpesvirus Raniid herpesvirus 2 (frog) Equid herpesvirus 1 Gill-associated virus Fowl plaque virus Cyprinid herpesvirus 3 (Goldfish) Bombyx mori cypovirus (Moth) Duck enteritis virus Norwegian salmonid alphavirus Hexaprotodon liberiensis gammaherpesvirus African swine fever Ictalurid herpesvirus 1 Canarypox virus Rachiplusia nu MNPV Anatid herpesvirus Ostreococcus virus Venezuelan equine encephalitis virus	Synechococcus phage (24) Prochlorococcus phage (10) Aeromonas phage (6) Acanthamoeba  polyphaga mimivirus (5) Rice gall dwarf virus (3) Cherry rasp leaf virus (3) Emiliania huxleyi virus (3) Cherry necrotic rusty mottle virus (2) Deftia phage (2) Turnip  mosaic virus Rice stripe virus (2) Plum bark necrosis stem pitting-associated virus (2) Aphid lethal paralysis virus (2) Cherry necrotic rusty mottle virus (2) Physalis mottle virus (2) Paramecium bursaria Chlorella virus (2) Broad bean wilt virus (2) Lettuce infectious yellows virus (2) Cycas necrotic stunt virus (2) Acanthocystis turfacea Chlorella virus (2) Cassava brown streak virus (2) Acute bee paralysis virus (2) Pennisetum mosaic virus (2) Sphaeropsis sapinea RNA virus (2) Choristoneura fumiferana nucleopolyhedrovirus (2) Anticarsia gemmatalis nucleopolyhedrovirus (2) Asian prunis virus (1) Microplitis demolitor bracovirus Trichoplusia ni ascovirus Sulfolobus virus Gordonia terrae phage White clover mosaic virus Alfalfa mosaic virus Pepino mosaic virus Bean pod mottle virus Turnip ringspot virus Eimeria brunetti RNA virus Pyrococcus abyssi virus Citrus yellow mosaic virus Patchouli mild mosaic virus Sunflower chlorotic mottle virus Brochothrix phage Rhodocococcus phage Rhizobium phage Xestia c-nigrum granulovirus Helicoverpa armigera granulovirus  Tomato leaf curl New Delhi virus Wheat eqlid mosaic virus Sonchus yellow net virus Grapevine chrome mosaic virus Actinoplanes phage  Feline immunodeficiency virus Spodoptera exiguae ascovirus Heliotis virescens ascovirus Hyphantria cunea nucleopolyhedrovirus Impatiens necrotic spot virus Xanthomonas phage Southern cowpea mosaic virus Sugarcane yellow leaf virus Cydia pomonella granulovirus Peanut mottle virus Rhodothermus phage

 

 

Table 2: Human vatches identical to those in Synechococcus phage or Epstein-Barr virus proteins: Genes that have been associated with multiple sclerosis in at least one study from MsGene are indicated or barred if negative. Autoantigens in multiple sclerosis are noted or highlighted in grey. The six highest scoring antigenic amino acids (B-cell: P,G,D,E,S,T) are highlighted in grey.+ = an amino acid with similar physicochemical properties. (See supplementary file msants.htm for antigen references)

 

Myelin related	Vatches
	Synechococcus phage	Epstein-Barr
CLDN11  claudin 11 (oligodendrocyte protein) MSGENE	CRALM & I+LLLTV & TIVSFG	ILLALC & LLTVLP
CNP  2',3'-cyclic nucleotide 3' phosphodiesterase MSGENE	DFLPLY &  KSYSK	SRGEEVG+ & SSETLR
MAG  myelin associated glycoprotein MSGENE	SWKP   & GTMVAV & CLCVVK & PAVSPED & IVCYIT	LLGDLGL & HFVPTR & YAEIRV
MBP  myelin basic protein MSGENE	LSPPK & NRIRQ & VPPRS	LSRFSW & SQGKGRG &  RTPPP
MOBP  myelin-associated oligodendrocyte basic protein MSGENE	PRSERQ & RLSKNQ	SICKSG & +WICC
MOG myelin oligodendrocyte glycoprotein MSGENE	CSFLLLL & LPVLLLQ	RPPF-RV & LRGKLR
MPZ  myelin protein zero MSGENE Also MPZL1  MPZL2 and MPZL3 (not shown) and MYEF2  myelin expression factor 2 MSGENE (not shown)	DPRW  GS VI &  PK SVRRR	PPDIVG & PGAPS
MYT1  myelin transcription factor 1 MSGENE Also MYT1L (not shown)	EN+EEIK &  LN STRCW & TPGCDG & PSSSY	LSTRCWE & NQDPE-KD
OMG  oligodendrocyte myelin glycoprotein MSGENE	SFLLLL & LNLSYN	NNIK-LD & S VTQP-VTK
PLP1  proteolipid protein 1 MSGENE	QDYEY  & TLVSLL F	FPGKVC & LVSLLT
PMP2  peripheral myelin protein 2 MSGENE   Also PMP22 (not shown)	WKLVS & IITIR	SIVTL+ & M-SNKFL
RTN4 Reticulon 4 MSGENE myelin derived neurite growth inhibitor (110) (also RNN4IP1, RTN4R, RNN4RL1 (not shown)	LSLKN & IDHYLG & SPSAIFS	SFRIYK &  KLVKE
Translation initiation (EIF2B1,2,3,4 and 5) only EIF2B1 is shown
EIF2B1  eukaryotic translation initiation factor 2B, subunit 1 alpha	TLL—FL-RDKG & SVAVSSG-E & TILTH	LLEFLK & HPWVD & ADLCH-F
Vitamin D related
VDR  vitamin D (1,25- dihydroxyvitamin D3) receptor	PGVQD & QTYI	GVQDA & EA+QDR
GC  group-specific component (vitamin D binding protein)	KLVNK & +VNKH	KRVLVL
CYP24A1  cytochrome P450, family 1,25-dihydroxyvitamin D(3) 24-hydroxylase	EDNFED & ESMRLT	Q+AAALP-PT & KLLKE
CYP27B1  cytochrome P450, family 27, subfamily B, polypeptide 1 25-hydroxyvitamin D-1 alpha hydroxylase	EHRRCR & LVRDV	RV-HRVRW &  RRC-QRAC
Other autoantigens (CLDN11, CNP, MAG, MBP see above)
ARRB2  arrestin, beta 2	LDK--YYHGE L+ & AAPET-+VPV+ & VYTITP & DTNLIE	PRPPTRL & PNPPR-P &  PVP –PRPP & PRPQS-PE
ATP2C1  ATPase, Ca++ transporting, type 2C, member 1	L+ FWREL-DN & KKLPIV & SKLVPP & LTQQQR	V+VDGDVV & SQ+-AVAIAS & EVSHRR &  VGIIDP
CRYAB  crystallin, alpha B	PLTI-SSL & PPSFLR &  FISREF & TSTSLS	SL--KDAVRVD G & RSPSP & ELRER & PVSNGG
CNTN2  contactin 2 (axonal)	LRDLL & FDNES & +SPRDN &  NGRR+P	PP—PRRPPG & HLTPTL-L &  PP PRRP-G—SW & RASPP T
HMOX1  heme oxygenase (decycling) 1	LHRK--LEQ LEEEIE & PGLRQ & WQEVI & YLGDLS	TR-SQAPP & SPSR-PGLR & DQSPS & LRQRAS
NEFL  neurofilament, light polypeptide	VRRSYSS & KDEVSE & LLVLRQ & AAISN+L &  AELLVL & SGSL+P &  APVSSS	EGLEE L & TQEKAQ & SLSVR S & SAPVS
NFASC  neurofascin	HTIQQK—FTLK & AKDPRV & SSSME &    +FRSGG	PDRP-DLE & GN+SSEAT & APP-LPP & GNPAPS & PPGLP
TALDO1  transaldolase 1	ISPFVGR & Y ELVEE & LISPFV & LVPVLS	PGRVS & PGRV-T & DARLS & +QIKNA+KL
SOD1  superoxide dismutase 1, soluble	GNAGS & GVIGT	VKVWGS+K & LGKGGN & EFGDN &  GRTLVV +
TKT Transketolase	YKSQDP & CLL-DGE &  LVAILD & EELCK	SLG+GLG & G+EDKE & GQAKHQ & LCGS-CG
TPI1  triosephosphate isomerase 1	FVGGNWK &  MA-EDGEEA & EVVCA P & EVVCA P	+GRKQSLG & TRP+PSP & LLG-LGPS & SLGPS-GR
Autoantigens: Tested:  ARRB2, ATP2C1, CNP, CNTN2, CLDN11 , CRYAB, HMOX1, MBP, MAG, MOG,  NEFL, NFASC PLP1, PMP2, SOD1,TALDO1, TKT PMP2 TALDO1, TPI1 . Not tested : ARRB1, ARR3,Bullous pemphigoid antigen, Beta amyloid , DKFZp686A1481, Enolase, Heat shock proteins, HSP60, HSP70, heterogeneous nuclear ribonucleoprotein B1, KIAA1279 lymphocyte protein p542  MOBP, OSP GAPDH, , PACSIN2, SPAG16,  Proteasomal components PSMA1, PSMA2

 

 

Table 3: Human proteins with significant overall homology to Epstein-Barr viral proteins: Viral translated genome vs. human proteins: No filter
Gene	P Value	Notes (from Entrez Gene)
IL10 interleukin 10 Identity : 140/175 (80%), Positives = 144/175 (83%), Gaps = 21/175 (12%) :=	4.00E-100	Cytokine produced by monocytes and lymphocytes. Affects in immunoregulation and inflammation. Down-regulates the expression of Th1 cytokines, MHC class II antigens , and costimulatory molecules on macrophages. Enhances B cell survival, proliferation, and antibody production.
POLD1 polymerase (DNA directed), delta 1, catalytic subunit 125kDa	1.00E-50	Involved in DNA replication and repair.
UNG uracil-DNA glycosylase	1.00E-32	Prevents mutagenesis by eliminating uracil from DNA molecules by cleaving the N-glycosylic bond and initiating the base-excision repair pathway
PFAS phosphoribosylformylglycinamidine synthase	2.00E-12	Catalyzes the fourth step of inosine monophosphate biosynthesis.
PRNP prion protein	2.00E-12	Membrane glycosylphosphatidylinositol-anchored glycoprotein that tends to aggregate into rod-like structures.
TBC1D9 TBC1 domain family, member 9 (with GRAM domain)	7.00E-07	??
RRM2 ribonucleotide reductase M2	4.00E-06	Catalyzes the formation of deoxyribonucleotides from ribonucleotides.
hCG23833-	4.00E-06	??
RRM1 ribonucleotide reductase M1	8.00E-05	Essential for the production of deoxyribonucleotides prior to DNA synthesis in S phase of dividing cells.
REV3L DNA polymerase zeta catalytic subunit	6.00E-04	Catalyzes the polymerization of deoxyribonucleotides into a DNA strand.
POLA1 polymerase (DNA directed), alpha 1, catalytic subunit	0.003	Encodes the catalytic subunit of DNA polymerase.
RRM2B ribonucleotide reductase M2 B (TP53 inducible)	0.005	Catalyzes the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, necessary for DNA synthesis.
The alignment of IL10 with the BPRF viral protein (capsid docking protein on nuclear lamina) IL10      SRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA 98 BPRF    SRVKTFFQTKDEVDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA 91 IL10      ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNK 148 BPRF    ENQDPEAKDHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQIKNAFNK 141 IL10      LQEKGIYKAMSEFDIFINYIEAYMTMKIRN 178 BPRF    LQEKGIYKAMSEFDIFINYIEAYMTIKAR- 170

 

 

 

 

 

 

 

 

 

Table 4 Human proteins with significant overall homology to Synechococcus phage proteins

 

 

Gene	P Value	Notes (from Entrez Gene)
G6PD glucose-6-phosphate dehydrogenase	1.00E-41	Generates  NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions.
H6PD hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)	4.00E-31	There are 2 forms of glucose-6-phosphate dehydrogenase. This form shows activity with other hexose-6-phosphates, especially galactose-6-phosphate.
RFC5 replication factor C (activator 1) 5, 36.5kDa	2.00E-18	Elongation of primed DNA templates by DNA polymerases requires the accessory proteins proliferating cell nuclear antigen and replication factors.
RFC2 replication factor C (activator 1) 2, 40kDa	4.00E-17	This gene encodes the 40 kD subunit, which has been shown to be responsible for binding ATP.
RFC4 replication factor C (activator 1) 4, 37kDa	2.00E-16	This gene encodes the 37 kD subunit.
PGD phosphogluconate dehydrogenase	1.00E-09	6-phosphogluconate dehydrogenase is the second dehydrogenase in the pentose phosphate shunt.
RFC3 replication factor C (activator 1) 3, 38kDa	6.00E-05	This gene encodes the 38 kDa subunit

 

 

Fig 1: Viruses expressing vatches identical to those in human multiple sclerosis autoantigens. The number of hits is influenced by the data; For example, MAG was homologous to Influenza proteins within multiple strains of the virus, occluding underlying data. Targeted sweeps, eliminating the first set of viruses would reveal a further set of with diminishing vatch size, but increased numbers. For example, the CNP vatches are smaller, but more viruses are implicated. Viruses implicated in multiple sclerosis (risk factors, modifying relapse, DNA or antibody detection) or demyelinating viruses are highlighted in grey as are the phage hosts similarly implicated. Bovine, ovine, feline and canine viruses are also highlighted.

CNP	Acanthamoeba polyphaga mimivirus Acinetobacter phage Aeromonas phage African swine fever virus Agrotis segetum granulovirus Ambystoma tigrinum virus Asian prunus virus Autographa californica nucleopolyhedrovirus Bacillus phage Bacillus thuringiensis phage Bathycoccus sp bovine viral diarrhea virus Bear Canyon virus Beet necrotic yellow vein virus Blackberry virus Y Borna disease virus Bovine herpesvirus 5  Bovine leukemia virus Bovine parainfluenza virus Bovine polyomavirus Buggy Creek virus Butterbur mosaic virus Canine calicivirus  Canine distemper virus Canine papillomavirus Cherry green ring mottle virusClostridium phage Cowpox Cyanophage PSS2  Duck hepatitis A virus Edge Hill virus Epizootic haematopoietic necrosis virus  Feline calicivirus  Feline immunodeficiency virus Gallid herpesvirus 3 Garlic virus Grapevine rupestris stem pitting-associated virus Helleborus net necrosis virus  Heliothis virescens ascovirus Hepatitis delta virus Horseradish latent virus  Human adenovirus Human astrovirus  Human coxsackievirus Human echovirus  Human enterovirus  Human herpesvirus 3 Human herpesvirus 5 Human herpesvirus 6B Human immunodeficiency virus 1 Human bocavirus Human parainfluenza virus Human parechovirus Human parvovirus  Human rhinovirus Hyperthermophilic Archaeal Virus Influenza A virus JC polyomavirusKashmir bee virus Klebsiella phage Lactobacillus phage  Leucania separata nuclear polyhedrosis virus Maize chlorotic dwarf virus Micromonas pusilla reovirus Mopeia Lassa reassortant 29 Mopeia virus Morogoro virus Murid herpesvirus 2 Mycobacterium phage Narcissus symptomless virus Nicotiana velutina mosaic virus Ovine adenovirus Paramecium bursaria Chlorella virus Peanut clump virus Peste-des-petits-ruminants virus Phaius virus  Porcine teschovirus Potato virus Pseudoalteromonas phage  Pseudocowpox virus Pseudomonas phage Prochlorococcus phage Ralstonia phage Rachiplusia ou MNPV  Rhodothermus phageSemliki forest virus Shallot virusShigella phage Simian picornavirus Spinach severe curly top virus Soybean mosaic virus Staphylococcus phage Synechococcus phage  Tiger frog virus Toscana virus Trichoplusia ni ascovirus Vibrio phage Watermelon mosaic virus Wood mouse herpesvirus  Zygocactus virus
MAG	Adult diarrheal rotavirus  Allium virus Bacillus phage  Burkholderia phage Colorado tick fever virus   Corynebacterium phage Enterobacteria phage Epstein-Barr virus Euproctis pseudoconspersa nucleopolyhedrovirus  Human herpesvirus 5  Influenza A virus  Lily virus X Marseillevirus  Musca domestica salivary gland hypertrophy virus Mycobacterium phage  Papillomavirus Pelargonium zonate spot virus Pseudomonas phage  Psittacid herpesvirus Staphylococcus phage Streptococcus phage A  Vibrio phage Yersinia phage
MBP	Bacillus phage Banna virus  Bean yellow disorder virus Broome virus  Campylobacter phage  Citrus tristeza virus Enterobacteria phage  Epstein-Barr   Felid Herpesvirus 1 Hepatitis B  Hepatitis delta Human adenovirus 4 Human herpesvirus 2 Human herpesvirus 3 Human herpesvirus 7 Human parvovirus Human T-lymphotropic virus  Influenza A Listeria phage Marseillevirus   Measles virus  Newcastle disease virus Norovirus Pepino mosaic virus  Rabbit hemorrhagic disease virus Rice stripe virus Rotavirus A Rubella virus Sapovirus sea trout rhabdovirus   Theiler's encephalomyelitis virus Torque teno virus   Yersinia phage
MOG	Avian orthoreovirus  Bacillus phage  Beet western yellows virus Border disease virus  Citrus tristeza virus Cypovirus Emiliania huxleyi virus  Epstein-Barr  Feline infectious peritonitis virus Gallid herpesvirus 2  Heliothis armigera cypovirus Hepatitis delta Human astrovirus  Human coxsackievirus Human immunodeficiency virus Human papillomavirus   Influenza A Louping ill virus Marseillevirus  Merino Walk virus Mokola virus   Mumps virus  Murine hepatitis virus   Mycobacterium phage Nemesia ring necrosis  Nyamanini virus Phocoena spinipinnis  Seoul virus Streptococcus pyogenes phage Temperate phage  Turnip mosaic virus  Watermelon mosaic virus  Yokose virus
EIF2B5	Acanthamoeba polyphaga mimivirus  Acidianus two-tailed virus  Actinoplanes phage Alfalfa mosaic virus  Ateline herpesvirus 3  Bacillus phage  Bacillus thuringiensis phage Bean common mosaic necrosis virus Burkholderia phage Canarypox virus Canine papillomavirus Citrus psorosis virus Clanis bilineata nucleopolyhedrosis virus  Clostridium phage  Corynebacterium phage Cyprinid herpesvirus 3  Ectocarpus siliculosus virus 1  Enterobacteria phage Enterococcus phage Epstein-Barr Escherichia phage  Equid herpesvirus 4 Equid herpesvirus 9 Equine rhinitis B Felid herpesvirus 1  Glossina pallidipes salivary gland hypertrophy virus  Haemophilus phage Haloarcula phage  Helicoverpa armigera NPV Hepatitis C Human coronavirus Human enterovirus  Human herpesvirus 1  Human herpesvirus 3 Human herpesvirus 5 Human herpesvirus 6 Human herpesvirus 7  Human herpesvirus 8 Human metapneumovirus Human papillomavirus  Hyperthermophilic Archaeal Virus  Klebsiella phage  Lactobacillus phage  virus Lactococcus phage  Latino virus Listeria phage Lymphocystis disease virus  Marseillevirus Mumps virus  Mycobacterium phage Norwalk virus  Oryza rufipogon endornavirus Ostreococcus virus Ostreococcus tauri virus 1 Plutella xylostella granulovirus Poliovirus Rachiplusia ou MNPV Ralstonia phage Rubella  SARS coronavirus  Shrimp white spot syndrome virus Soybean chlorotic mottle virus Spinach latent virus St. Louis encephalitis virus Streptococcus phage Suid herpesvirus 1  Taterapox virus Tupaiid herpesvirus Yersinia phage virus Trichoplusia ni SNPV s  Staphylococcus phage Shigella phage Variola West Nile virus

 

Fig 2 Human proteins containing Epstein-Barr or Synechococcus phage vatches : Those in boxes have been reported as multiple sclerosis susceptibility genes. Grey-shaded proteins have been reported as autoantigens. For the chi squared test, the expected number of genes is (328/35000)*N: For the autoantigens the expected number of autoantigens is (37/35000)*N, where 328 is the number of recorded Msgenes, 37 the recorded number of autoantigens, and 35,000 the number of human proteins.

Epstein Barr virus (no filter) N=132 MSgenes = 1 Autoantigens = 0	ABCA7ABCB11 ACACB ADAM2 ADNP2 GALT ANXA6 ATE1 BCL2L12BRAPBRD4 DST KAZ C2CD3 CACNA1D CC2D1B CCDC90A CDA CDKL5 CEACAM16 CEP135 CIT COL18A1 CRIPAK CUX1 DDX31 DKK2 DNAJC14 DRPLA EGLN2 ELN EMID1 EIF4G3 EXO3CL2 FAM120C FAM8A1 FBLN2 GPR124 GPR128 GPRIN1 GRM7 HK3 HMX1 HNRNPUL1 HTR3C IER3IMMT IGH@ ZCCHC24 INS-IGF2 IQCK IRAK1 KANK2 LAG3 MA MAN1A1 MAVS MCM3AP MED15 MEOX1 MHC class I antigen MLLT1 MON1B MPEPL1 MSLN MTCH1 MTSS1 MUC12 MUC16 MUC2 MUC5B NRARPNUDT14 ODF3B PARVA PCD10 PMP22 PKD2 PLCE1 PLEKHA7 PLEKHG4 PLEKHG5 PLEKHH1 PLXNB3 PRDM16 QSOX2 RAB3IP RAI1 RASA4 RASSF7 RBM25 REEP1 RGL3 RGMB RPGRIP1L RRM1 RRM2 RRM2B SAV1 SF1 SLC6A5 SMPDL3A SOCS7 SPIB SPRR2D SRRM3 STAB2 SYNGR2 TBC1D9 TMEM132A TOM1 TPRN TRIP6 TSC2 TSPAN3 TTC19 TWF1 UNG STXBP5L WIZ WNK1 WNK2 XIRP1 ZAP70 ZC3H18 ZMYM3
Epstein Barr virus (filter = multiple sclerosis) N=204 MSgenes = 55 Autoantigens = 4	ADAD1 ADAMTS14 ADAMTSL1 ADNP ALK ALS2 ICAM3 RAD54L APEX2 ASAP1 BANK1 BDNF BICD1 BRCA2 IRF5(IRF5) BSV BUD13 C1orf125 C4A C4orf33 C6orf27 CACNG4 CASP8  CBLN2 CCNH CD2 CD200R1 CD247 CD2AP CD58 CD6 CD97 CENPC1 CEP170 CHRND CIITA RAD51 CLDN11 CLEC16A CNP CNTF CNTN2CTGF CYFIP2 CYP24A1 CYP27B1 CYP4V2 DARS2 DCP1A ECE2 EIF2B4EME1 EN1 PLCE1 ENTPD1 EPC1 ERCC1 ERCC3 ERCC8 ERVWE1 EVI5 FEN1 FH TLR3 FLNA FMN2 FURIN GABBR1 GBP1 GDF5 GPC5 GFI1 GRN GH6PD HAVCR2 HDAC11 HELZ HLA-DRA HS3ST2 HSPB2 HTATIP2 IFIH1 IFNAR1 IFNGR2 IL16 IL22RA1 LEKR1 IL7R IGH@ IRF9 JARID2 KANK1 KCNB2 DCLRE1C KCNJ7 KIF1A KIF21B KLRB1 LAMB2 LIG3 LILRA3 LMNB1 MATN3 MBP MERTK MMP25MMP28 MMP9 MMS19 MNAT1 MPG MPHOSPH9 MUS81 MUTYH MX1L MYO9B METTL1 NBN NCKAP5 NDUFS5 NEIL2 NFASC NHEJ1 NRLP11 NTHL1  KLC1 OAS3 OGG1 OLIG1 OPN3 OPTN PAM PARD3B PDZRN4 PARP2 PRKDC PCSK5 PDE4B PDMB9 PGGT1B PKN1 POL3RC POLD1 OLIG3 POLE POLG POU2AF1 PREP PSMD2 PTPRZ1 RAD23A RAD51C RAD51L3 RAD52 RAD54B RFC1 RGMA RGNEF RNF19A RPA1 RPL19 SCHIP1 SH2B3 SH2D2A SHROOM3 SLC25A27 SLC25A36 SLC44A4 SMAD7 SMUG1 SNPH SPARCL1 SPG11 SYN3 TLR3 TMEM39A TRA@ TRD@ TRB@ TKT TNFSF12 TRIM15 TSC2 USP31 VAV2 VPS54 WDR64 WDR7 WT1 LINGO1 XAB2 XRCC1 XRCC2 XRCC3 XRCC4 ZIC1ZP4
Synechococcus phage : No filter N = 232 MS Genes =9 Autoantigens = 6	ABCA4 ABCG2 ADAMTS12 AEBP1 AGPAT9 AHDC1 ALDH8A1 AMICA1ANKRD12 ANP32E ATRN BAI2 C1orf94 C20orf72 CATSPER1 CCDC46 CCDC146 CCNK CD1B CDH12 CD2AP CELSR3 CENPI  CLCN6 CPEB4 CPN2 CROT CRYAA CSPG4 CTNNBL1CYP2R1 CYP4V2 DCAF13 DERL2 DGKB DHX36 DMRTB1 DNAJC14 DNAJC16 DOLPP1 DYM EIF3M EP400 ESPN FAM38B FAM72A FAM72D FBXO38 FIBP FIP1L1 FKBP9 FLJ22184 FMN1 FMOD FOLH1 FOXO3 FOXP1 G6PD GP1BAGOLGB1GPR85 GPR98 GRHPR GTPBP6HAO2 HEATR5B HEATR7B2 HLA-DQB1 HLA-DRB1 H6PD HSF2BP HSP90AA1 IFI44 IGH@ INPP5D JAKMIP3 KIAA1826 KIF2A KIF18A KIF23 KIR3DL2 KLKB1KRTAP19-3 KRT13 LAMA1 LINGO2 LOC285074 LOC401097 LOC727787 LOC100288144 LOC100509054 LOC100293516 LOC100508689 MACF1 MAFG MAGEC1MAG MAPKBP1 MAP4K2 MAPK9 MAPK10 MBP MCM6 MDN1 MEGF11 MGAT4A MGAT5B MLL MLXIP MMP21 MOG MPP5 MTMR7 MTRNR2L3 MTMR7 MUC2 MUC16 MYT1 MYT1L MBTPS2 MYO15A  MYO18A NCKAP1 NCL NEXN NFASC NIPBL  NOX1 NPSR1 NRIP1 OBSCN OR5M9 OR5K3 OR5K4 OR14L1P OXCT1PAPPA2 PCNXL2 PEX1 PFKFB1 PGAP1 PHRF1PGD PKD1POLA1 POLD1PRRT1   PSMC3 PTPN14 RFC1 RFC2 RFC3 RFC5 RFXP3 RMND5A ROBO1RORB RSPH10B RSPH10B2 RREB1 RRM1 RSAD2 RXFP3 SAMD1 SARNP SCN7A SERPINI2 SF3A1SGCZ SLC2A8 SLC6A9 SLC22A8    SLC35E3 SLC38A11 SLC44A4  SMC1A SMC1B SMC4 SPATA5 SRGAP2P2 SSH1 STRC SYNE1 SYN2 SYNM TAF1 TAF9 TALDO1TAOK2 TDRD3 TEAD3 TFE3 DAPK1 TMEM41B TMEM41B  TMEM88B TMEM117 TMEM168 TMEM202   TNIK TRA@ TRB@ TRD@ TRIM34 TTN TLL1TSPAN12 TULP1 UBA2 UBE2J1UHRF1BP1 VWCE WDHD1  WDR81XKR3 XKRX YRDC ZNF148  ZNF587 ZNF770
Synechococcus phage query multiple sclerosis N= 213 genes MS genes = 41 Autoantigens = 5	ADAD1 ADARB1 ADAMTSL1 ADAMTS14ADNP APEX2 ASAP1ASAP2 ASPN BANK1 BSN BUD13 CNLB CASP10 CCDC46 CCL14 CD40 CD2AP CD200R1 CD226 CDK7 CEP170 C4A CHRND CLDN11 CLEC16A C1orf125 C20orf46 CKB CDC42 CD97 CNTF CPAMD8 CYFIP2 CYP2R1 CYP27B1 CYP4D2DDX58 DBP DCP1A  DCLRE1C EIF2B2 EIF2B3 EMR2 ENTPD1EPC1 ERCC3 ERVWE1 EME1 EVI5 FAM69A FLAD1 FLNA GABBR1 GAPDH GBP1 GDF5 HAPLN1 HAVCR2 HDAC11 HELZ HLA-DRA HLA-DRB1HLA-G HTATIP2 H6PD ICAM3 IDS IFNAR1 IFT74 IL16 IL21R IL22RA1 JARID2 KANK1 KCNB2 KCNH7 KCNIP1 KCNK5 KIF1A KIF1B KIF5A KIF21B LANB2 LEKR1 LIG4 LINGO1 MGAT5 MBD4 MMP8 MMP28 MMS19 MNAT1 MPHOPH9 MRE11A MUS81 MXI1 MX2 MYO9B NCKAP1 NCKAP5 NFASC NFKBIL1 NIPA1 NLRP11 NOD1 OAS1 OAS3 OPTN OR2H1 PADI2 PARD3B PARP2 PDE8A PDLIM7 PDZRN4 PGGT1B PLD5 PLKCE1 PMN2 PNKP POLE POLD1 POLG PON2 PREP PRKDC PSMD2 PTPRC PTPRZ PNPT1 PVALB PVRL2 RAD21L1 RAD23A RAD23B RAD54B RAD54L RFC2 RFC4 RFC5 RGNEF RGS7 RNF19A RPA1 S1PR1 S1PR3 SLC25A27 SLC44A4 SMUG1SNPH SPG11 ST8SIA1 TAC4 TALDO1 TKT TLR7 TLR8 TLR10 TNFRSF19 TRIM26 TSC1 TSC2 VAV2 CIITA OLIG3   USP31 VPS54 POLB WDR7 WDR64 WRN XAB2 XRCC5 XRCC6 ZIC1 ZNF45 ZNF433   ALS2   KCNA5
No Filter		Epstein-Barr	Synechococcus phage
Fold enrichment genes		0.8 (NS)	3.68: Chi2 =22.5 p<0.0001
Fold enrichment Autoantigens		0 NS	24.4: Chi2=135 p < 1e-30
Filter = multiple sclerosis		Epstein-Barr	Synechococcus phage
Fold enrichment genes		28.8: Chi2 =1418 p < 1E-30	20.5: Chi2 =767 p<1E-30
Fold enrichment autoantigens		18.2: Chi2= p = 0.0026	21.8: Chi2=16.1 p= 0.00006

 

 

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