key: cord-0888073-2g5uxglk
authors: Donati, Donatella
title: Viral infections and multiple sclerosis
date: 2020-03-01
journal: Drug Discov Today Dis Models
DOI: 10.1016/j.ddmod.2020.02.003
sha: faa52b8b14c7d1b1034774ef0e2c54c3a4041c68
doc_id: 888073
cord_uid: 2g5uxglk

The etiology and pathogenesis of MS is likely to involve multiple factors interacting with each other, and the role of infectious and viral agents is still under debate, however a consistent amount of studies suggests that some viruses are associated with the disease. The strongest documentation has come from the detection of viral nucleic acid or antigen or of an anti-viral antibody response in MS patients. A further step for the study of the mechanism viruses might be involved in can be made using in vitro and in vivo models. While in vitro models, based on glial and neural cell lines from various sources are widely used, in vivo animal models present challenges. Indeed neurotropic animal viruses are currently used to study demyelination in well-established models, but animal models of demyelination by human virus infection have only recently been developed, using animal gammaherpesviruses closely related to Epstein Barr virus (EBV), or using marmosets expressing the specific viral receptor for Human Herpesvirus 6 (HHV-6). The present review will illustrate the main potential mechanisms of MS pathogenesis possibly associated with viral infections and viruses currently used to study demyelination in animal models. Then the viruses most strongly linked with MS will be discussed, in the perspective that more than one virus might have a role, with varying degrees of interaction, contributing to MS heterogeneity.

. Some aspects of its etiology and pathogenesis are still unclear, however it is generally accepted that MS is an inflammatory disease with autoimmune characteristics influenced by environmental or infectious factors in genetically susceptible individuals. These factors are supposed to interplay to varying extent, contributing to the heterogeneity of MS [2] .

The typical MS lesions are multiple perivascular white matter plaques of demyelination associated with various degrees of inflammatory cells [3, 4] . Additionally diffuse neurodegeneration and plaque-like demyelination has also been described in the deep and cortical grey matter [5] [6] [7] [8] .

The possible involvement of viral agents has been suggested by the detection of viral nucleic acid, protein or antiviral antibodies in the blood, CSF or brain tissue, and several viruses have been specifically associated with MS, though a definitive cause-effect relationship has not been demonstrated. Virus infections might contribute to MS through different mechanisms in various combinations [9, 10] . These are direct toxicity, molecular mimicry, dual T cell receptor, bystander activation and epitope spreading, briefly outlined below.

Though clinical and experimental evidence accounts for an autoimmune pathogenetic mechanism for MS development, the presence of a direct viral toxic mechanism on actively infected neural cells cannot be ruled out, on the basis of detection of cellular damage not related to inflammation or autoimmunity. Indeed oligodendrocytic dystrophy without IgG and complement deposition, as if a direct damage would occur, has been found in some MS plaques [3] , and primary defective oligodendrocyte precursor cells contributing to blood-brain barrier (BBB) dysfunction and disease perpetuation have recently been demonstrated [11] .

Molecular mimicry is the mechanism by which an immune cross-reaction towards myelin components can be induced by homologous viral sequences [12] or structurally similar peptides [13] through presentation by major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) to autoreactive CD4 + T cells. A number of viruses have been demonstrated to induce disease at least in part through this mechanism, as in the case of HSVinduced stromal cheratitis [14] and Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) [15] .

The possibility that a T cell can carry more than one TCR, contrasting with previous assumption, has been demonstrated [16] , suggesting that some T cells might carry two different combinations of TCR, specific for both myelin and viral antigens respectively and upon activation would react against both antigens [17] .

Bystander activation implies that a viral infection can elicit an over-reactive inflammatory response due to tissue damage and consequent unveiling and presentation by APCs of hidden autoantigens and production of novel autoreactive T cells and plasma cells [18] .

The mechanisms illustrated above, damaging myelin-producing cells, would cause release of myelin fragments in the inflammatory environment, triggering a self-sustained breakdown of myelin, with additional recognition of further epitopes [17] . This process, known as epitope spreading, was first described by Lehman et al. [19] and has been studied in TMEV-IDD [15] .

Several models of demyelination are known [20, 21] . In vitro models based on glial cell lines include the use of oligodendrocyte [22, 23] , astrocyte [24, 25] and microglial [26, 27] cell lines [28] and progenitor-derived glial cells [29] [30] [31] [32] [33] . In vivo models include the most extensively studied animal model for MS, experimental autoimmune encephalomyelitis (EAE), obtained by immunization with myelin proteins with adjuvants, or passive transfer of autoreactive T cells with or without pathogenic auto-antibodies in susceptible animals [34] [35] [36] [37] .Various clinical types of EAE can be induced in rodents and primates (marmosets and rhesus macaques), ranging from acute monophasic [38] to a chronic course, either relapsing/remitting [39] or chronic progressing [40] . EAE can also develop after induction of toxic agents, such as cuprizone [41] , lysophosphatidilcholine [42] , ethidium bromide [43] . EAE can also occur spontaneously in transgenic mice expressing a T-cell receptor specific for myelin oligodendrocyte glycoprotein (MOG) [44] .

The strains of alphavirus SFV commonly used are the mutant variant M9, highly virulent, and the avirulent A7 strain. This latter strain has been more widely studied and can cause an acute secondary demyelination with encephalomyelitis after viral clearance in B6 and, in a milder form, SJL mice [45] . During the demyelinating phase the virus is not found in the CNS and a molecular mimicry between viral surface glycoprotein E2 and MOG peptide 8-32 has been demonstrated, with production of cross-reactive T cells and antibodies [46] . Additionally, SFV induces the expression of adhesion molecules, among which VLA-4 [47] , in the CNS.

The coronavirus MHV can infect humans, mice, rats and nonhuman primates [48] . After intracranial inoculation of susceptible mice, MHV induces an acute severe encephalomyelitis infecting oligodendrocytes, astrocytes and microglia. After two weeks of infection the viral loads in the surviving animals are undetectable, but oligodendroglial expression of viral antigen is detected, accounting for viral persistence, with the development of a chronic immune-mediated demyelinating disease [49] . Infiltrating T cells and macrophages are thought to be the main effectors of demyelination, with a possible additional role for a direct toxic effect due to viral persistence in oligodendrocytes. Interestingly, in chronic demyelinating lesions endogenous remyelination has been documented [50] . Since remyelination can occur also in MS [51] , MHV can be considered as a valid model to study specific aspects of MS pathogenesis.

The demyelinating disease induced by TMEV, a natural enteric pathogen of mice, can be considered the most relevant virusinduced animal model of MS. TMEV-IDD has interesting similarities with MS, since the virus establishes a persistent infection resembling the chronically progressive form of MS, with myelin reactive T cells and anti-myelin antibodies. Furthermore, mice with TMEV-IDD can undergo spontaneous remyelination with partial recovery of neurological functions, as observed in MS [52] . These features refer to the TO TMEV subgroup, including the DA and BeAn strains. The other viral subgroup, namely GDVII, includes the strains GDVII and FA and is extremely virulent, causing death within 2 weeks after infection. Infection of SJL/J mice is characterized by an acute phase, between day 3 and 10, in which viral antigens and genome are found in neurons [53] , followed by a decrease of the viral load, with incomplete clearance. A chronic phase then ensues 1 month after inoculation, with viral persistence in oligodendrocytes, astrocytes and microglia/macrophages [54] . The subsequent immune reaction does not eliminate the virus, but leads to a chronic inflammation, driven by virus-specific CD4 + T cell response, self-sustained by epitope spreading [15] , with immune response against myelin proteins [55] .

Japanese macaque encephalomyelitis (JME), a spontaneous inflammatory demyelinating disease similar to MS, was first reported in 2011 in a colony of Japanese macaques and associated to a novel simian herpesvirus, JMERV [56] . Viral genomic characterization revealed that JMERV is a gamma-2 herpesvirus closely related to rhesus macaque rhadinovirus and human herpesvirus 8 [57] . Immunopathological analysis documented the presence of several key signatures associated with MS, such as CNS-infiltrating T cells with Th1 and Th17 phenotypes besides MRI similarities, suggesting a potential use as an MS model [58] .

Though no MS-specific virus has been found so far, investigation in this area has continued based on epidemiological and laboratory data [9, 59] . Due to the complexity and heterogeneity of MS it is possible that more than one viral agent is involved [60] . Additionally, as well as commensal bacteria, ubiquitous viruses composing a common flora of viruses, named virome, might challenge and shape the immune system similarly and/or complementarily to bacterial common flora [61, 62] .

The prevalence of EBV seropositivity in the world's population is approximately 95% [63] , while almost 100% of MS patients are seropositive [64] . Moreover, a history of infectious mononucleosis significantly increases the risk of MS [65] and CSF-restricted EBV-specific oligoclonal bands (OCB) in a subset of MS patients has been detected [66, 67] though the specificity of such result is still under debate [68] . Contrary to the unequivocal serological data, the search of EBV in MS brain has generated conflicting results, which were studied by the NeuroproMiSe EBV Working Group with the final conclusion that an unequivocal proof of EBV CNS infection in MS is lacking [69] . On this basis it has been hypothesized that EBV would rescue ''forbidden'' memory B cells directed against a CNS epitope [70] . To explain the inconsistent detection of EBV in MS lesions, it has been suggested that EBV memory B cells would lose the episomic EBV DNA upon replication, but would retain the ''forbidden'' epitope recognition, possibly activating a molecular mimicry mechanism [71] . Additionally, a ''two hit hypothesis'' has been formulated to explain the association of EBV infection with MS: during primary infection EBV would disrupt BBB permeability, allowing activated immune cells to enter the CNS thus generating a cascade of events leading to CNS inflammation [72, 73] .

Animal models for EBV infections use mice infected with murine gammaherpesvirus 68, a mouse pathogen, which upon induction show an accelerated course of EAE [74] , or marmosets naturally infected with the gamma1-herpesvirus callitrichine herpesvirus 3 (CalHV3) which, similarly to EBV, infects and immortalizes B cells [75] . It has been demonstrated that CalHV3-infected B cells can act as antigen presenting cells (APCs) not only via MHC class II molecules to CD4 T cells, but also via MHC class I molecules to CD8 T cells [76] . Using this model, a conversion from a tolerogenic destructive processing of MOG peptide to productive processing and autoaggressive T cell activation was demonstrated [77].

Very interesting results come from a recent study showing that MS patient sera, differently from healthy controls, recognize unique EB nuclear antigen (EBNA) 411-426 epitopes with antibodies cross-reacting with myelin basic protein (MBP). Moreover SJL/J and Balb/c mice injected with EBNA 411-426 peptide develop signs of EAE [78] . Based on these findings, a new line of investigation can be developed for a further insight into the possible role of EBV through molecular mimicry in MS pathogenesis.

An association of HHV-6 with MS has been suggested since 1993 [79] and has focused a wide number of studies [80] . Two HHV-6 species are known, HHV-6 A and HHV-6 B, sharing a 95% homology [81] . HHB-6 B accounts for most symptomatic infections during infancy, including exanthema subitum (or roseola infantum), after which it can establish latency. The prevalence of HHV-6A (currently not associated with a distinctive disease) is not known due to the cross-reactivity of HHV-6A and HHV-6 B antibodies, however early studies documented an HHV -6A higher detection rate in the CSF than in PBMCs [82] . Viral DNA persists in episomes but can also integrate into host cell chromosomes, with a prevalence of approximately 0.85% in the general population [83] , and neural cells can be a site of latency.

A considerable number of studies, summarized by Virtanen and Jacobson [9] , have demonstrated an association between HHV-6 and MS, either by direct DNA detection in MS lesions or increased antiviral antibody titres in MS patients though other investigators did not confirm this association [84] . Interestingly, a sequence homology between HHV-6 protein U24 and myelin basic protein [85] and cross-reactivity of autoreactive T-cells with MBP [86] has been demonstrated, suggesting a molecular mimicry mechanism.

For cellular entry HHV-6 uses the complement regulatory receptor CD46 [87] , which is expressed in adult oligodendrocytes, astrocytes and microglial cells [88] , accounting for viral neurotropism. While CD46 is missing in rodents, it is expressed in the common marmoset (Callithrix jacchus), which can be used as a model not only for MS by EAE induction [89] , but also for the study of HHV-6 infection [90] . Very promising results were obtained in EAE marmosets previously infected intranasally with HHV-6, which showed a significant accelerated EAE compared to uninfected animals [91] . Moreover HHV-6 antigen expression was upregulated in EAE lesions, similarly to what observed in MS [92, 93] and a decrease of naïve CD8 cells with a peripheral expansion of effector/memory CD8+ cells correlating with EAE duration in HHV-6/EAE marmoset was documented. In the light of these findings, the authors concluded that HHV-6 might prime the immune system, creating a ''fertile field'' for the expansion of autoaggressive T cells, then triggered by other environmental factors [94] .

HERVs were incorporated in the human genome millions of years ago and the presence and/or activation of three HERVs (HERV H, HERV K and HERV W), has been associated with MS [95, 96] .

The findings would suggest that HERV activation might trigger a demyelination process, contributing to MS progression [97] . HERVs can be activated by several simuli, including viruses such as VZV, HSV-1, EBV and HHV-6 [98] , however the most widely studied association is with EBV. Indeed the HERV W MS-associated retrovirus (MSRV) activation during infectious mononucleosis and the notion of the high prevalence of EBV positivity in MS would suggest that MSRV activation might possibly act as an effector in MS, being triggered by EBV latent infection [99] . Again this viral interaction would be in agreement with the theory of a ''fertile field'' [94] , created in this case by a previous EBV infection.

It has been demonstrated that injection of a MOG peptide in an emulsion containing MRSV Env protein instead of complete Freund adjuvant can induce EAE in mice, providing a model to study the role of MSRV in MS [100] .

It has been demonstrated that the intrathecal polyspecific humoral response of MS patients has Measles, Rubella and VZ viruses (MRV) as its most frequent component [101] , providing a useful help for MS diagnosis. Moreover VZV infection is associated with higher risk of developing MS [102] . However appropriate animal models for the study of a role in MS are lacking, despite some attempts for the study of VZV neurotropism [103] .

In conclusion, the role of viral infections in MS has still to be defined, and the possibility that more than one virus is involved in MS pathogenesis should be taken into account. Moreover the possible degrees of interaction between viruses and other infections or environmental and genetic factors might vary to a great extent, consistently with MS heterogeneity. The detection of viral components in MS lesions or antiviral immune response in MS patients associated with clinical relapses are highly suggestive of a role for viruses, possibly either as triggers or co-factors in the development of the disease. However the ubiquity and the specificity for humans of these viruses make it difficult to study possible mechanisms. For this reason the establishment of new models of MS in infected animals is very promising, and provides an important tool to shed light into the definition of viral role in MS.

I have no conflict of interest.

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