key: cord-0034992-z4h5pxj2
authors: Williams, Elizabeth L.; Jacobson, Steven
title: Viral Infection and Multiple Sclerosis
date: 2007
journal: Immune Regulation and Immunotherapy in Autoimmune Disease
DOI: 10.1007/978-0-387-36003-4_11
sha: 9f8e0eb203d7ed8f1a72cdc100b2a8b12093c356
doc_id: 34992
cord_uid: z4h5pxj2

nan

Studies during the 1960s were the first to discover that persistent viral infections can cause chronic neurological disease (Gilden, 2005) . Since that time, many viruses have been positively associated with numerous neurological and chronic diseases. The characterization of virus in affected tissues, increased risk of disease in immunosuppressed patients, and response of patients to antiviral therapy all point investigators toward an infectious etiology of the disease. Multiple viruses have been shown to produce demyelination, and a temporal association of postinfectious encephalomyelitis has been shown after smallpox vaccination and measles, varicella, or rubella infection. Viruses are capable of establishing persistent, latent infection in host organisms leading to continuous viral replication over time without killing the host. Reactivation of latent virus can produce clinical disease, although the stimulus of reactivation has yet to be characterized.

The association of viruses with numerous neurological disorders in animals and humans has led to the theory that multiple sclerosis (MS) may also be the result of infectious or virus-triggered neuropathology (Gilden, 2005) . One such example is the correlation of JC virus with progressive multifocal leukoencephalopathy (PML), the only human demyelinating disease with a proven viral cause. Another example is the temporal association of viruses with postinfectious encephalomyelitis, a demyelinating disorder that occurs as a complication of either smallpox vaccination or measles infection. Theiler's murine encephalomyelitis virus (TMEV) leading to central nervous system (CNS) demyelination is another example that supports a role for virus in MS. Although a viral association with MS may be the result of primary infection, more likely it is the reactivation of virus years after the primary infection that leads to the development of MS, similar to JC virus in PML and TMEV, both of which result from reactivation of latent virus in the CNS that may lyse oligodendrocytes, initiating an immunopathological process that leads to demyelination (Gilden, 2005) .

Although the etiology of MS is still unknown, much research has been done to gain a better understanding of the factors involved in the development of disease. Epidemiological studies have supported both a genetic component and an environmental trigger in MS. These environmental triggers have been suggested to be transmissible agents such as a viral or bacterial infection. Although the immunology of MS is complex, further support for infectious triggers is indicated by the role of Th1-type CD4+ cells in disease pathogenesis. Th1 cells dominate viral infections and allow for the proliferation of CD8+ cells. In addition, studies have demonstrated increased CD8+ cytotoxic T cells in MS lesions, which suggests a role for these effector T cells in the pathogenesis of this disorder. The observation of increased immunoglobulin G (IgG) responses in MS further supports a role for infection in disease, as most diseases that show high concentrations of IgG are inflammatory and infectious in nature. The involvement of viral infections in MS coupled with the long-held view of MS as an autoimmune disease suggests that molecular mimicry may play a role in MS disease pathogenesis, as studies have found that myelin basic protein (MBP)-specific T cells can be effectively activated by MBP homologue from numerous viruses. This review highlights a number of these diverse observations that lend support to a viral etiology in MS.

Multiple sclerosis is a demyelinating disease affecting the CNS, with its onset occurring between 20 and 40 years of age. The pathological marker of disease is white matter lesions or plaques resulting from loss of axonal myelination due to inflammation, with noticeable lymphocyte infiltration and oligodendrocyte loss (Ffrench-Constant, 1994) . Lesions are characterized by demyelination, edema, and disruption of the blood-brain barrier (Hemmer et al., 2002) . Symptoms of disease begin with recurring inflammatory attacks involving significant neurological impairment, ranging from vision problems and difficulty walking to paralysis (Steinman and Zamvil, 2003) . Although the cause of MS is unknown, it has typically been considered a CD4+ Th1 cell-mediated disease. Disease begins when the blood-brain barrier is breached, and activated immune cells attack components of the myelin sheath insulating axons. Epidemiological studies have suggested both a genetic and environmental component in MS, leaving open the possibility of a viral trigger in MS.

The prevalence of MS in specific ethnic groups residing in the same environment supports a role for genetic susceptibility (Haines et al., 1998) . The familial aggregation of disease, observed in population and family studies, shows an increased risk in first-, second-, and third-degree relatives over the general population (Dyment et al., 2004) . Adoption studies show an increased risk of developing MS only in biologically related individuals (Ebers et al., 1995) . In twin studies, monozygotic twins have a higher concordance rate than dizygotic twins (Haines et al., 1998) . Although the monozygotic twin concordance rate of 20% to 30% indicates that genetics can predispose an individual to MS, nongenetic factors such as environmental triggers and immune responses must play a role in determining the overall etiology of disease (Dyment et al., 2004) . The genetic component most often observed is the HLA-DR2 allele on chromosome 6p21, and this gene could contribute 10% to 60% of the genetic risk factor (Haines et al., 1998) . Allelic variants of chromosomal regions are linked to increased disease risk, and these MHC genes could predispose disease through thymic selection or presentation of antigen to T cells, or both (Ermann and Fathman, 2001) .

Multiple sclerosis affects approximately 1 million people worldwide, although it is much more prevalent in Caucasians, and 350,000 of people affected reside in North America (Steinman, 2001a) . A geographical gradient of north to south has been found, with the Northern Hemisphere having an increased prevalence of disease. The geographical distribution is not related to genetics alone, as the prevalence of disease in Caucasians who migrate outside of Europe or North America is one-half that of those living in many parts of the Northern Hemisphere. Migration studies found that the geographical risk is acquired by the age of 15, as migration from an area of high risk to one of low risk during adolescence confers a reduced risk of developing disease. One explanation for the geographical distribution observed is the reduced sunlight exposure at higher latitudes, as ultraviolet (UV) radiation may exert an effect on vitamin D or cause an excess of melatonin, which enhances Th1 responses (Sospedra and Martin, 2005) .

Twice as many women as men develop MS, a phenomenon observed with other autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis (Steinman, 2001a) . This finding suggests a role for hormones as risk factors in MS and is supported by the finding that relapse rates are decreased during pregnancy and increase afterward. Additionally, disease often worsens during menstruation; high estradiol and low progesterone have been correlated with increased magnetic resonance imaging (MRI) disease activity; and estriol has shown a therapeutic effect in RR-MS (Sospedra and Martin, 2005) . The mechanism of hormonal action is unknown, although the stimulatory effect of estrogen on proinflammatory cytokine secretion is a likely mechanism.

The mechanisms of action for the observed geographical distribution could be numerous, with the "hygiene hypothesis" frequently mentioned as a possible mechanism. The "hygiene hypothesis" states that less hygienic environments, which predispose children to infections, helps protect against later disease by driving the immune system toward Th1 responsiveness. In contrast, children in a more hygienic environment with low exposure to infectious disease, as is the case in developed countries, tend toward Th2 responsiveness, and these are the children who are thought to be more prone to developing atopic allergic disease. Although this hypothesis is difficult to substantiate, it elucidates a possible mechanism as to the increased prevalence of disease in the Northern Hemisphere.

Interestingly, epidemics of MS have been observed, suggesting that a transmissible agent may be associated with disease pathogenesis. The most well known epidemic is that of the Farøe Islands during World War II, where MS was unknown until 1940 when British soldiers arrived and an epidemic broke out shortly thereafter (Kurtzke, 2000) . Prospective studies have also shown that MS relapses often follow viral infections, and seasonal variation in the incidence of new MS cases exists. Experimental autoimmune encephalomyelitis (EAE) studies have also suggested a transmissible agent, as almost 100% of transgenic mice expressing T cell receptors (TCRs) specific for an encephalitogenic peptide of MBP develop EAE when housed in non-pathogen-free conditions, whereas those in pathogen-free conditions do not (Goverman et al., 1993) . Additionally, common viral infections are temporally associated with MS exacerbations in some patients (Sibley et al., 1985) . Many groups have searched for bacteria and viruses that may be associated with disease, and to date about 20 organisms have been associated, although none has gained acceptance as the causal agent of MS (Table 11 .1).

Although the etiology of MS is unknown, epidemiological studies point to a role for both genetics and environmental triggers in the development of disease. Figure 11 .1 depicts a visualization of how these overlapping criteria may combine to lead to autoimmune disease.

Like many other chronic inflammatory diseases, MS has traditionally been thought to be a CD4+ Th1-mediated disease. Naive T cells differentiate into Th1 cells in the presence of interferon-γ (IFNγ) and interleukin-12 (IL-12) and into Th2 cells in the presence of IL-4 and/or IL-6. The presence of Th1-or Th2-type cytokines also function to inhibit the generation of the other response. Th1 cells dominate viral and bacterial infections owing to IL-12 (produced by dendritic cells and macrophages) and IFNγ, produced by natural killer (NK) cells and CD8+ T cells. The two subsets of CD4+ cells have very different functions: Th1 (Huseby et al., 2001; Steinman, 2001b) . Previous to this, the focus of EAE research was on the role of myelin-specific CD4+ T cells because of their ability to induce EAE predominantly and lead to cytokine activation similar to that seen in MS (Steinman, 2001b) . Additionally, lesions in EAE induced by CD8+ CTLs for MBP closely resemble MS lesions and exhibit extensive demyelination, indicating that CD8+ CTLs are capable of damaging MHC I-expressing oligodendrocytes (Huseby et al., 2001) . CD8+ CTLs are the primary effector T cells that lyse virus-infected cells by recognizing short peptide fragments (usually nine amino acids in length) in association with HLA class I molecules (Neumann et al., 2002; Shresta et al., 1998; Trapani et al., 2000) . They are associated with antigen-specific apoptosis in the CNS, as their specificity for virus-infected cells prevents widespread tissue damage (Barry and Bleackley, 2002; Russell and Ley, 2002) . CTLs kill target cells by a number of mechanisms, including the Ca 2+ -dependent release of lytic granules and the Ca 2+ -independent binding of Fas to Fas-L on the CTLs leading to the activation of caspases (Barry et al., 2000; Green et al., 2003; Medana et al., 2000) . CD8+ CTLs accumulate around lesions in infectious and autoimmune neurological disorders including MS (Neumann et al., 2002) . It has come to be appreciated that MHC I-restricted CD8+ CTLs have been found to outnumber CD4+ T cells in MS lesions almost 10:1 (Booss et al., 1983 ). CD8+ CTLs have been shown to be enriched at the site of actively demyelinating lesions in MS (Monteiro et al., 1995) and the clonal proliferation of CD8+ CTL cells has been observed to be much greater than that of CD4+ cells in the T cell infiltrate in inflammatory lesions (Babbe et al., 2000) . CD8+ CTL responses to MHC I peptides of MBP are elevated in MS patients compared to healthy individuals, suggesting that CTLs specific for MBP may become activated in MS patients (Crawford et al., 2004) . Neuronal cells do not constitutively express the MHC I molecule necessary for CD8+ activation (Neumann et al., 2002) , but invading CD8+ CTLs release proinflammatory cytokines including IFN-γ, tumor necrosis factor-α (TNFα), and TNFβ (Medana et al., 2000; Woodland and Dutton, 2003) . IFNγ inhibits viral replication, activates macrophages, and induces the express of MHC I molecules on glial cells and neurons (Vass and Lassmann, 1990) .

Recognition of the involvement of CTLs in the pathogenesis of MS is suggestive of a role for virus in lesions, as CD8+ CTLs are implicated in the direct killing of resident cells during infection. The preponderance of CTLs in inflammatory lesions supports a role for CTLs as important effectors in MS, and further study of these cells is crucial to our understanding of their involvement in inflammation and MS.

The response of IgG to autoantigens on cell surfaces leads to the rapid destruction of these cells and inflammatory injury. About 90% of MS patients have high concentrations of IgG in brain and cerebrospinal fluid (CSF), further suggesting an infectious etiology for MS. Other diseases that show high concentrations of IgG are inflammatory and most are infectious (Gilden et al., 1996) . Attempts to bind IgG extracted from MS brain and CSF to that of healthy controls and MS patients has been relatively unsuccessful, and points to a possible flaw in the hypothesis of autoantigen involvement in MS (Gilden, 2005) . Research on IgG oligoclonal bands from other studies have suggested that oligoclonal IgG in MS is antibody-directed against the infectious cause of MS (Swanborg et al., 2003) .

Viral infections could lead to autoimmune diseases, including MS, via three possible mechanisms: molecular mimicry, bystander activation, and viral persistence. Each of these mechanisms involves the interaction of the virus with the immune system, "priming" the host for subsequent immunopathology by initiating the immunoreactivity that leads to autoimmune disease (Fujinami et al., 2006) . In molecular mimicry, T cells react in the periphery to foreign molecules with enough similarity to self-antigen, causing the T cells to become activated and allowing them to cross the blood-brain barrier (Wucherpfennig and Strominger, 1995) . Resting autoreactive T cells cannot cross the blood-brain barrier and therefore must be activated in the periphery. Many microbial proteins share sequence homologies with components of the myelin sheath, most notably myelin oligodendroglial glycoprotein (MOG), MBP, and proteolipid protein (PLP); and this homology allows activated T cells to attack myelin (Steinman, 2001a) .

Viruses have been shown to cross-react with host proteins, activating MOG-or MBP-specific T cells (Fujinami et al, 2006; Guggenmos et al., 2004) . MBP-specific T cells can be effectively activated by MBP homologues from various viruses, including herpes simplex, human papillomavirus, adenovirus type 12, and Epstein-Barr virus (Wucherpfennig and Strominger, 1995) . Cross-reaction between viruses and hosts, although common, must occur in a disease-related epitope for autoimmune disease to occur; and this epitope must include peptides that can be presented by MHC II molecules on antigen-presenting cells (APCs) to CD4+ cells (Fujinami et al., 2006) . Viruses such as those of the herpesvirus family are good candidates for molecular mimics, as they can establish latent and persistent infection; and chronic infection may permit constant antigenic stimulation of autoreactive T cell clones (Wucherpfennig and Strominger, 1995) . Persistent viral infections could damage the immune system as a result of the continual presence of viral antigen (Fujinami et al., 2006) .

Although many pieces of evidence point toward a role for viral infection in the development of MS, making a positive association between a virus and neurological disorders such as MS is challenging, especially when the infection may be ubiquitous in the population. A number of chronic human illnesses have been suggested to be "triggered" by microorganisms, and proving the infectious etiology of these diseases has required extensive support from multiple sources (Carbone, 2005) . The traditional rules for proof of pathogenicity, Koch's postulates, are often not practical when examining viral triggers, as it is rare that any single line of evidence is sufficient to prove an infectious nature of chronic disease. Proof is often realized only with the accumulation of sufficient and supportive data by multiple sources and techniques. Although it is extremely important to detect the suspected pathogen in diseased tissue, detection does not necessarily prove causality, as the organism could be an innocent bystander or endogenous flora-present but not a factor in the disease. Additionally, a lack of detection does not necessarily mean an association does not occur, as the method used may not be sensitive enough to detect low levels of virus. Any association must include evidence from multiple sources, including genetic, epidemiological, microbiological, and pathological components (Carbone et al., 2005) .

Animal models can be helpful in generating hypotheses and can be useful to support viral associations with disease, but proving a link between an organism and a human chronic illness in an animal model again does not prove causality. Therefore animal models cannot always be used to draw conclusions regarding viral association. Obviously, viral associations in human disease would be more compelling, using methods such as the polymerase chain reaction (PCR) to detect viral nucleic acid in human tissue or by measuring antibody levels in serum, plasma, or CSF. Often, prospective and retrospective serological studies are used to help elucidate the cause of chronic illness; but here too lie problems, as evidence of past exposure does not imply current infection, seronegative individuals could have past infection, antibody levels could decline over time, and often tests are not sensitive enough to detect low levels of virus present in chronic infection.

A viral trigger of disease is any virus that sets into motion or expedites the disease process and can be involved in the development of disease in a number of ways, including persistence as a chronic infection and induction of destructive host immune responses in genetically susceptible individuals (Carbone et al., 2005) .

The large body of evidence that points to a role for viral infection(s) in MS pathogenesis has led to numerous possible viral candidates for association with this disease. Viral triggers have been suggested to be involved in MS for more than 100 years (Marie, 1884) , and since then numerous viruses have been suggested as causative agents in the pathogenesis of MS (Table 11 .1). Some viruses that were initially thought to be associated with MS have, upon further analysis, shown less likelihood of association. This long-list of viruses can be interpreted in one of three ways: (1) viruses have nothing to do with MS pathogenesis, as none of these observations have stood the test of time;

(2) the list is incomplete and the "MS virus" has yet to be discovered; (3) some but not all of these viruses are associated with disease in a subset of MS patients. This review argues for the latter interpretation by focusing on three ubiquitous candidate agents: human endogenous retroviruses and the herpesviruses Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6), whose association with MS is currently being extensively explored.

Retroviruses were named for the presence of the viral enzyme reverse transcriptase (RT), an RNA-dependent DNA polymerase; the assessment of RT activity is the main criterion for distinguishing retroviruses. Most human endogenous retroviruses (HERVs) are endogenized exogenous retroviruses; they were incorporated into our genome during primate evolution and now make up 8% of the human genome. Additionally, retroviruses have been implicated in immunodeficiency and neurological disorders, and RNA for HERVs may be upregulated during inflammation (Christensen, 2005) . As HERVs are ubiquitous, their pathogenicity must be modulated by genetic and environmental factors. The diversity of HERVs in the human genome is the result of homologous recombinations, as seen in the MHC region (Christensen, 2005) .

Several human endogenous retroviruses have been implicated in MS based on the presence of activated HERVs in MS blood, increased RNA expression of HERV in brain tissue from MS patients, and elevated levels of HERV antibodies in the serum and CSF of MS patients (Brudek et al., 2004) . Some studies have found that MS patients have increased concentrations of antibody to RT from HERVs compared to control patients, although other studies have had contradictory findings (Gilden, 2005) . Retrovirus-like particles (RVLPs) have been found in T cell cultures from patients with MS, and cultures also express EB virusencoded proteins (Haahr et al., 1991) . Multiple sclerosis-related virus (MSRV, previously known as LM7), a HERV, was first isolated from MS patients, and its expression has been associated with disease severity and duration, although increased expression has also been observed in other inflammatory neurological diseases (Dolei et al., 2002; Zawada et al., 2003) . It is unknown if HERVs are a causal factor in MSl; and if not causal they could be involved as a synergistic player, an immune activator or suppressor; alternatively, HERVs could be an epiphenomenon (Christensen, 2005) . HERVs or HERV-activated proteins could act as insertional mutagens, as regulators of gene expression, or they could interact with exogenous viral RNA or elicit abnormalities as viruses.

HERVs could also be transactivated by human herpesviruses, as it has been found that herpesviruses and retroviruses are multiplicatively synergistic in stimulating cell-mediated immune responses (Brudek et al., 2004) . The combination of HERV-H and either HHV-6 or HSV-1 increased cell proliferation in MS patient cell cultures over that of healthy controls, although the increase was not significant (Brudek et al., 2004) . Regardless of the significance, the cell-mediated immune response of HERV and herpesvirus antigens in conjunction has a stimulatory effect (Christensen., 2005) . The interaction of HERVs and herpesviruses is similar to situations found in vivo, as HERVs are ubiquitous in the genome and herpesviruses are highly prevalent throughout the human population; therefore an interaction between them is plausible. The herpesviruses could also function to induce aberrant expression of HERVs, as it has been found that the retroviruses human immunodeficiency virus (HIV) and human lymphtropic virus (HTLV) in the presence of herpesviruses encode transactivating factors that can exacerbate disease (Christensen, 2005) . It is clear that further studies are needed to better elucidate the role of HERVs in the development of MS.

Epstein-Barr virus (EBV) is a herpesvirus believed to infect 95% of the world's population. EBV is trophic for B cells and can establish latency and persist throughout life. Primary infection can be asymptomatic or develop into infectious mononucleosis (IM), which is associated with lymphoproliferation in an immunocompromised host.

Whether EBV has a role in MS has yet to be elucidated, but virtually all patients with MS have antibodies against EBV versus 86% to 95% of healthy individuals (Gilden, 2005) . Individuals who had IM in the past have an increased risk of developing MS (Lindberg et al, 1991) . Prospective samples from MS patients have been shown to be positive for EBV antigens EBNA-1 (Epstein-Barr nuclear antigen-1) and VCA (viral capsid antigen); high activity to EBNA-1 significantly increased the risk for MS, whereas VCA was not associated with MS development (Sundstrom et al, 2004) . Antibodies to EBNA-1 have been found in 85% of MS patients compared to only 13% of EBV-seropositive controls (Bray et al, 1992) . Increases in serum titers of EBV antibodies have been found before the onset of MS in prospective studies, with the strongest predictor of MS the serum levels of IgG to EBNA-1, supporting a role for EBV as a risk factor in MS (Ascherio et al., 2001; Levin et al., 2005) . While oligoclonal IgG bands in brain and CSF of MS patients have been well documented, but there is no indication that these bands are directed against EBV (Gilden, 2005) .

Although many studies have pointed toward a role of EBV in MS, EBV RNA and DNA have not been found in MS brain tissue (Challoner et al., 1995; Hilton et al., 1994; Morre et al., 2001; Sanders et al., 1996) or in CSF of MS patients (Martin et al., 1997; Morre et al., 2001) , and studies examining EBV DNA in blood and serum have produced mixed results. EBV could lead to MS though molecular mimicry, as EBNA-1 has been shown to have homologies with MBP, and EBV peptides may activate HLA-DR2-restricted MBP-specific T cells (Sundstrom et al., 2004) .

Human herpesvirus-6 was first isolated in 1986 from immunosuppressed patients with lymphoproliferative disorders and HIV infection (Salahuddin et al., 1986) . HHV-6 is classified as a hematotropic or beta-herpesvirus, along with cytomegalovirus (CMV) and human herpesvirus-7 (HHV-7); HHV-6 bears 67% sequence homology with CMV (Lawrence et al., 1990) . HHV-6 infects most individuals between 6 and 12 months of age (Okuno et al., 1989) , and more than 90% of the general population is seropositive for HHV-6 (Kimberlin, 1998) . Primary HHV-6 infection has been identified as the causative agent in exanthum subitum (roseola infantum), a febrile illness sometimes resulting in seizures and neurological complications including meningitis and encephalitis Yamanishi et al., 1988; Yoshikawa and Asano, 2000) .

Two variants of the virus have been identified, variant A and variant B, exhibiting a nucleotide sequence homology of between 88% and 96%. HHV-6A has been implicated in multiple sclerosis and is associated with viral persistence and reactivation in the CNS Hall et al, 1998) . The HHV-6B variant is primarily associated with symptomatic infections during infancy and limbic encephalitis and is the variant implicated in exanthem subitum. HHV-6B appears to be the predominant strain of the virus, as it is detected more frequently, although the 6A variant has been suggested to have a greater neurotropic potential than 6B (Dewhurst et al., 1993; McCullers et al., 1995; Wainwright et al., 2001) .

After primary infection, HHV-6 can establish a lifelong latent infection, with the viral genome persisting in peripheral blood mononuclear cells (PBMCs) and in the salivary glands (Campadelli-Fiume et al., 1999) . HHV-6 DNA has been observed in the CSF of children not only during primary infection but also subsequent to infection (Caserta et al., 1994) . Reactivation of latent virus is often seen in immunocompromised patients, as half of all patients who undergo stem cell or bone marrow transplant develop active HHV-6 infection (Caserta et al., 2001; Singh et al., 2000) and may contribute to disease in HIV infection and chronic fatigue syndrome. HHV-6 is suggested to be an immunosuppressive agent, although the mechanism of immunosuppression is unknown. Even in immunocompetent subjects HHV-6 has been shown to invade the CNS directly, creating a persistent, latent infection (Saito et al., 1995) .

HHV-6 has been indicated as a cofactor in neurological diseases, most notably demyelinating diseases including multiple sclerosis (Challoner et al., 1995; Soldan et al., 1997) . Progressive multifocal leukoencephalopathy (PML), another demyelinating disease of the CNS, is generally thought to be caused by reactivation of the human polyoma virus, JC virus, although it is now suggested that HHV-6 activation, in conjunction with the JC virus, is associated with demyelination in PML (Mock et al., 1999) . In addition to demyelinating diseases, HHV-6 has also been implicated as a cause of epilepsy, encephalitis, encephalomyelitis, and meningitis. HHV-6 has been shown to infect numerous CNS cell types, including T cells, oligodendrocytes, and astrocytes, and has been shown to infect primary human fetal astrocytes in vitro (Albright et al., 1998; He et al., 1996; Lusso et al., 1988) . HHV-6 DNA has been detected in CSF of patients with limbic encephalitis, and astrocytes positive for HHV-6B DNA were the most commonly infected cell type in the hippocampus (Wainwright et al., 2001) .

The case for HHV-6 as one of many potential viral triggers in MS is supported by a large number of studies that are consistent with the following observations: First, the initial HHV-6 infection during infancy is compatible with the epidemiology of MS that suggests exposure to a microbial agent during childhood. Second, HHV-6 has been shown to infect numerous cell types in the CNS, including lymphocytes and glial cells (He et al., 1996) . Third, herpesviruses are highly neurotropic and neuroinvasive, and they are often implicated in neurological diseases and other CNS complications (Wilborn et al., 1994) . Fourth, the high seroprevalence of HHV-6 throughout the world is consistent with the high prevalence of MS. Lastly, herpesviruses are able to establish persistent, latent infections in the CNS and can be reactivated as a result of stress or other infections, the same stressors that have also been associated with MS exacerbations. Although an association between HHV-6 and MS remains to be definitively proven, the large numbers of reports that demonstrate a role of this virus in MS pathogenesis (Fig. 11 .2) are compelling evidence that suggest that this may be an important area of investigation.

Supportive proof for an infectious etiology of disease can be realized only through the accumulation of data by multiple approaches. For example, the association of HHV-6 with MS has been made based on: (1) immunological detection: differential antibody and virus-specific lymphoproliferative responses between patients and controls; (2) molecular detection: demonstration of virus-specific DNA and RNA sequences in cell-free and cell-associated compartments;

(3) pathological detection: expression of viral antigen and/or viral DNA in affected brain tissue from diseased patients; (4) clinical and radiological correlations: temporal association of virus specific responses with clinical and MRI parameters of MS disease. This review highlights the findings from these diverse approaches that have been used to correlate HHV-6 infection and MS as an example of how an association of a ubiquitous virus with MS is currently being explored.

Viral associations in MS have been suggested for over 50 years with numerous representations from many viral families including both DNA and RNA viruses Cermelli and Jacobson, 2000; Johnson, 1994) largely based on increased antibody detection in patients versus controls. These studies typically have measured long-term IgG titers in either serum or CSF as a measure of past exposure. By contrast, a number of compelling studies (Ablashi et al., 1998 (Ablashi et al., , 2000 Friedman et al., 1999; Ongradi et al., 1999; Soldan et al., 1997; Villoslada et al., 2003) have demonstrated significant elevations of HHV-6-specific IgM in the serum of MS patients compared to patients with other neurological diseases, other inflammatory diseases, and healthy controls (Soldan et al., 1997) . Detection of an early antibody response (IgM) to an early antigen of HHV-6 (p41/38) has suggested that active HHV-6 replication may be occurring in a subset of MS patients. Consistent with these HHV-6-specific IgM responses detected in MS sera, other studies have found an increased percentage of MS patients with anti-HHV-6 IgM in CSF. For example, Ongradi et al. demonstrated anti-HHV-6 IgM in 57% of MS patients and 0% of controls (Ongradi et al., 1999) . In addition to IgM responses, increased IgG antibodies to HHV-6 has been one of the most consistent findings in most of the studies (Fig. 11 .2) examining the role of HHV-6 in MS serum (Ablashi et al., 1998 (Ablashi et al., , 2000 Caselli et al., 2002; Liedtke et al., 1995; Ongradi et al., 1999; Sola et al., 1993 Sola et al., , 1997 Wilborn et al., 1994) 

Immunological detection-positive correlation (Ablashi et al., 1998 (Ablashi et al., , 2000 Caselli et al., 2002; Derfuss et al., 2005; Friedman et al., 1999; Liedtke et al., 1995; Ongradi et al., 1999; Sola et al., 1993; Soldan et al., 1997 Soldan et al., , 2000 Tejada-Simon et al., 2002; Villoslada et al., 2003; Wilborn et al., 1994) . No correlation (Enbom et al., 1999; Nielsen et al., 1997; Xu et al., 2002) . Molecular detection-positive correlation (Ablashi et al., 1998; Akhyani et al., 2000; Alvarez-Lafuente et al., 2002b , 2004b Berti et al., 2002; Chapenko et al., 2003; Clark, 2004; Fillet et al., 1998; Goldberg et al., 1999; Liedtke et al., 1995; Rotola et al., 2004; Soldan et al., 1997; Tejada-Simon et al., 2002; Tomsone et al., 2001; Wilborn et al., 1994) .

No correlation (Al-Shammari et al., 2003; Martin et al., 1997; Mirandola et al., 1999; Taus et al., 2000) . (Ablashi et al., 1998; Derfuss et al., 2005; Ongradi et al., 1999) . Although some reports have failed to support these findings (Enbom et al., 1999; Nielsen et al., 1997; Taus et al., 2000; Xu et al., 2002) the wide variety of assay platforms, use of different viral preparations, and variations in selection of patients and controls make it difficult to compare results among studies. There is a clear need to define a "gold standard" for HHV-6-specific IgG and IgM responses in both serum and CSF to better characterize the antibody response to HHV-6 in MS. Immune responses to HHV-6 have also been investigated by examining virus-specific T cell proliferation. In a study from our own group, although there was no difference between MS patients and controls in response to HHV-6B or HHV-7, a significantly higher percentage of MS patients demonstrated proliferative responses to HHV-6A . Increased frequency of HHV-6A specific T cells in MS is of interest because the HHV-6A variant has been suggested to be more neurotropic (Hall et al., 1998) and to have a greater propensity for latency and reactivation (Dewhurst et al., 1993) , and HHV-6A sequences are more often detected in MS sera Alvarez-Lafuente et al., 2002b) and CSF (Rotola et al., 2004) than variant B. More recently, T cells recognizing the recombinant 101-kDa protein of HHV-6 that corresponds to an immunodominant region of the virus occurred at a significantly lower precursor frequency in MS patients than controls . These responses were associated with a skewed cytokine profile characterized by the inability to produce IL-4 and IL-10. The authors concluded that the diminished T cell response to HHV-6 and skewed Th2 cytokine profile was associated with ineffective clearance of HHV-6 in MS, suggesting a role for this virus in MS disease pathogenesis .

Although not universally accepted (Nielsen et al., 1997; Xu et al., 2002) , most reports employing immunological detection methods consistently demonstrate HHV-6-specific responses in MS patients (Fig. 11.2) . Collectively, these studies continue to support a role for HHV-6 as a reasonable candidate for an etiological agent in MS.

Detection of HHV-6 DNA and RNA sequences by primary and nested PCR has been used to demonstrate the presence of HHV-6 in MS patients compared to controls. The detection of HHV-6 DNA in cell-free compartments (i.e., serum, CSF, urine) has been suggested to reflect potentially active HHV-6 replication, whereas detection of cell-associated (e,g. PBMCs) HHV-6 sequences may not be able to distinguish latent from active virus. Moreover, using HHV-6 variant-specific primers and probes, it is possible to distinguish HHV-6A from HHV-6B infection. Recently, real-time quantitative PCR methods have been used to measure HHV-6 viral loads accurately from PBMCs and CSF lymphocytes; and RT-PCR has been used to amplify HHV-6 mRNA sequences. Demonstration of HHV-6-specific RNA in PBMCs is also suggestive of active HHV-6 replication (Alvarez-Lafuente et al., 2004b) .

The results from these molecular analyses support the HHV-6 immunological observations found in MS. In both serum and CSF, HHV-6 DNA has been found in significantly more MS patients than controls (Ablashi et al., 1998; Akhyani et al., 2000; Alvarez-Lafuente et al., 2002b; Berti et al., 2002; Chapenko et al., 2003; Clark, 2004; Fillet et al., 1998; Goldberg et al., 1999; Liedtke et al., 1995; Rotola et al., 2004; Soldan et al., 1997; Tejada-Simon et al., 2002; Tomsone et al., 2001; Wilborn et al., 1994) . We have demonstrated HHV-6 DNA in serum from approximately 25% of MS patients compared to 0% of controls including patients with other inflammatory diseases, other neurological diseases, and healthy subjects Berti et al., 2002; Soldan et al., 1997) . Although we find that most MS patients and controls have detectable HHV-6 DNA in the PBMCs, others have demonstrated HHV-6 DNA sequences in PBMCs more frequently in MS patients (Fig. 11 .3) (Chapenko et al., 2003; Tomsone et al., 2001) . Importantly, the increased frequency of detection in MS patients was HHV-6-specific, as no differences were observed between MS patients and controls to seven other human herpesviruses tested (Fig. 11.3 ).

More recently a number of studies have focused on HHV-6 RNA in PBMCs of MS patients. Alvarez-Lafuente and colleagues demonstrated HHV-6 mRNA for three immediate early (IE) genes by quantitative real-time RT-PCR in a substantial number of RRMS patients and not in healthy blood donors (Alvarez-Lafuente et al., 2002b) . As a method of distinguishing active from latent infection, this study compared mRNA expression of the IE genes U89/90, U16/17, and U94 with the expression of U94 alone. Presence of U94 in the absence of other IE gene transcripts has been associated with latent HHV-6 infection (Mirandola et al., 1998) . More MS patients were found to have mRNA for all three IE genes 202 Williams and Jacobson than U94 alone, indicating active HHV-6 infection in this subset of MS patients (Alvarez-Lafuente et al., 2002b) . No significant difference in U94 expression was found between MS patients and controls. Similar to the observations using immunological detection methods that suggest a role of HHV-6 in MS, most reports based on molecular methods support these findings (Fig. 11. 2), although with greater variability among these studies (Al-Shammari et al., 2003; Martin et al., 1997; Mirandola et al., 1999; Taus et al., 2000) . This is not unexpected as it is well appreciated in molecular PCR-based assays that there is considerable variability in the use of these methods. Different regions of the virus are amplified with different sets of primers and probes having varying degrees of sensitivity and specificity that are used in either primary or nested PCR conditions. Only through standardized PCR assays can these difficulties be overcome.

Detection of HHV-6 by immunological and molecular means are important observations in associating this virus with MS. However, the demonstration of this (or any infectious) agent in diseased MS brain material would be even more compelling. As access to MS brain tissue is limited, only a few studies demonstrating HHV-6 in MS brains have been reported. Indeed, the first report of an association of HHV-6 with MS was based on the immunohistochemical detection of HHV-6 antigen in oligodendrocytes from MS plaques (Challoner) . This landmark study using an unbiased subtractive hybridization approach (representational differential analysis) demonstrated HHV-6-specific sequences in MS plaque material compared to controls. A more recent report also localized HHV-6 to oligodendrocytes in MS brains (Opsahl and Kennedy, 2005) . Although HHV-6 mRNA was detected in both MS and control brain tissue, higher levels of HHV-6 viral activity as determined by percentage of HHV-6 mRNA-positive oligodendrocytes were demonstrated in MS patients compared to controls (Opsahl and Kennedy, 2005) . In this study, quantitatively more HHV-6 mRNA to both immediate early and late genes was detected in MS lesions versus controls, suggestive of an active HHV-6 infection (Opsahl and Kennedy, 2005) .

The demonstration of HHV-6 in MS brain is also supported by studies examining HHV-6 DNA in brain tissue by PCR. Cermelli et al., using laser microdissection, found statistically more HHV-6 DNA in active MS plaques than in normal-appearing white matter (NAWM) from the same MS patients and brain material from patients with other neurological diseases including inflammation (Cermelli et al., 2003) . These findings are consistent with previous studies that demonstrated HHV-6 DNA by PCR more often in brain tissue from MS patients than in controls (Friedman et al., 1999; Sanders et al., 1996) . In a case report of brain biopsies from five MS patients, all sections demonstrated high levels of HHV-6 DNA-positive cells by in situ PCR, most of which were oligodendrocytes (Goodman et al., 2003) . Although HHV-6 DNA was detected in oligodendrocytes, HHV-6 antigen was not, using immunohistochemical analysis for HHV-6 p41, p101, or gp116. However, HHV-6 antigen was detected in hypertrophic astrocytes to the HHV-6 gp116 protein staining in two of the five patients (Goodman et al., 2003) . The authors concluded that the prevalence of HHV-6 genome-containing cells in MS lesions support the hypothesis that HHV-6 plays a role in the demyelinative pathogenesis of MS. The demonstration of HHV-6 in pathological material is crucial to support the association of this agent in this disorder. Although studies using different antibodies have found HHV-6 antigen more often in MS patients than controls (Friedman et al., 1999; Knox et al., 2000; Virtanen et al., 2005) , others have not (Blumberg et al., 2000; Coates and Bell, 1998) . Clearly, more studies are needed to qualitatively and quantitatively detect both HHV-6 genome and HHV-6 antigen(s) in the CNS of MS patients.

Immunological, molecular, and pathological detection of HHV-6 infection in MS have supported an associative role of HHV-6 in this disease. Even more compelling are the clinical correlative studies between virus and MS disease development or progression. As most MS patients are clinically defined by relapses and remissions, a number of reports have investigated whether HHV-6 can be differentially detected during these phases of disease. A significant correlation was demonstrated between serum HHV-6 DNA and the number of MS patients with clinical exacerbations, diagnosed by patient complaints and neurological examination (Berti et al., 2002) . The detection of HHV-6 DNA more often in patient serum during exacerbation than remission suggests that active HHV-6 infection may play a role in the development and/or progression of MS (Berti et al., 2002) . These findings were supported by others who found a higher viral load of HHV-6 in MS patients during exacerbations than during remissions for three different IE HHV-6 genes: U89/90, U16/17, and U94 (Alvarez-Lafuente et al., 2004b) . Similarly, Chapenko et al. identified periods of HHV-6 viremia by detecting HHV-6 DNA in plasma only during periods of new MS activity (exacerbations), as indicated by the presence of gadolinium (Gd)-enhanced lesions, and not during periods of relative remission, as indicated by an absence of Gd-enhanced lesions (Chapenko et al., 2003) . The study concluded that the risk of exacerbation was 2.5 times greater in patients with active HHV-6 infection than in those with latent infection. We have also observed that serum HHV-6 levels cycle over time by a longitudinal analysis of monthly serum samples (24-month time course) (Berti et al., 2002) . As it is known that Gd-enhanced MRI lesions also cycle, more patients must be evaluated to determine if there is a correlation between HHV-6 and these lesions.

Of interest is the use of IFNβ, an established therapy for MS based on its ability to reduce the frequency and severity of exacerbations, disability, and brain lesions in patients with MS (IFNB, 1993) . IFNβ has become one of the most commonly used treatments for RRMS, and the success of IFNβ in MS is thought to be not only a result of its antiinflammatory properties but may also be due in part to its antiviral activity (Alvarez-Lafuente et al., 2004a; Hong et al., 2002) .

IFNβ has been demonstrated to inhibit significantly the viral replication of HHV-6, as treatment with IFNβ decreased the amount of HHV-6 DNA in the serum of MS patients as compared to untreated MS patients . In another study, IFNβ was not found to diminish HHV-6 DNA in the serum of MS patients during either relapse or remission (Alvarez-Lafuente et al., 2004a) . Although DNA in serum was not reduced, they did find that the viral load in the serum of patients undergoing an acute attack was significantly lower in the IFNβ RRMS group than in the untreated RRMS group (Alvarez-Lafuente et al., 2004a) .

Collectively, these clinically correlative studies, together with the immunological, molecular, and pathological detection of HHV-6 in MS, continue to support a role for the involvement of HHV-6 in this disorder.

Further research is needed to associate HHV-6 infection with the immunopathogenesis of MS definitively. Although more data are needed to make an association, it is clear from the breadth of research in the field that most studies find a positive correlation between infection and disease. However, more studies focusing on associations with HHV-6 and MS may get us no closer to proving a causal role for this agent. In all of the studies to date demonstrating the presence of HHV-6 in MS patients, it is difficult to conclude whether the virus is the "cause" of the disease or a mere bystander that results from other immunological events that nonspecifically reactivate the virus. Although some studies have controlled for this ( Fig. 11. 3), the argument of HHV-6 (or any infectious agent) as an epiphenomenon with little to do with the pathogenesis of MS is valid.

To address this crucial issue, a growing number of investigators believe that only through well controlled interventional clinical trials with effective and safe antiviral agents can a causal role be made for any infectious agent in MS. To date, only a handful of reports have attempted to intervene in MS with anti-beta herpesvirus drugs. Although no compound has been formally approved as an antiviral for the treatment of HHV-6 infection, antiviral agents used for CMV infection or other herpesvirus treatment, including ganciclovir, acyclovir, cidofovir, and foscarnet, are often used (De Bolle et al., 2005) . Antiviral drugs used for the treatment of herpesvirus infections act by targeting virus-specific kinases and inhibiting viral DNA polymerases (Bech et al., 2002) .

Several case studies of the successful use of ganciclovir for HHV-6 encephalitis in bone marrow transplant (BMT) patients have been published, and prophylactic therapy with ganciclovir has also been shown to be effective in preventing HHV-6 reactivation in BMT patients (Johnston et al., 1999; Mookerjee and Vogelsang, 1997; Rapaport et al., 2002; Rieux et al., 1998; Tokimasa et al., 2002; Wang et al., 1999; Yoshida et al., 2002) . Case studies of foscarnet for the treatment of HHV-6 encephalitis have been mixed, with some showing successful results (Bethge et al., 1999; Zerr et al., 2002 ) and others yielding less success (Rossi et al., 2001; Tiacci et al., 2000) . Clinical reports of cidofovir are more limited owing to the risk of nephrotoxicity; and one report found ganciclovir more successful than cidofovir in treating HHV-6 encephalomyelitis in an immunocompromised patient (Denes et al., 2004) .

There have been limited studies in the use of antivirals in MS. One study found that valacyclovir, the valine ester of acyclovir with increased bioavailability, did not reduce the formation of active lesions over the 24-week course of treatment (Bech et al., 2002) . However, a subgroup of MS patients with high disease activity, as measured by more than one active MRI lesion, valacyclovir was found to have reduced numbers of new active MRI lesions (Bech et al., 2002) . In a more recent clinical trial in MS patients, valacyclovir, though the results were not statistically significant, was found to have a stabilizing effect on clinical progression of disease (Friedman et al., 2005) .

These clinical trials serve to highlight the challenges in designing and interpreting an antiviral trial in MS. First, the choice of drug is critical, particularly with respect to HHV-6, as it is not clear what is the most effective anti-HHV-6 compound to use. Is the intent to interfere with HHV-6 replication in the periphery or CNS-and in which cell type (e.g., virus-infected lymphocytes or glial cells)? Second, which group of MS patients should one select? If HHV-6 plays a role in only a subset of patients, how is this group to be selected and what assay(s) should be used to monitor patients? Lastly, what clinical and/or radiological measures are to be used as a primary outcome measure of treatment efficacy? The MS trial design has made significant advances over the years with a number of drugs approved for the treatment and many more in the pipeline (McFarland and Reingold, 2005; Mouzaki et al., 2004) . If antiviral drugs are to be part of the armamentarium for MS, these drugs must be shown to interfere with virus growth or replication in patients with detectable levels of virus. If these criteria are met, coupled with clinical and/or radiological improvement with antiviral therapy, there can be confidence for an etiological role of a virus in MS.

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Human herpesvirus-6 (HHV-6) infection in multiple sclerosis: a preliminary report

Tissue distribution and variant characterization of human herpesvirus (HHV)-6: increased prevalence of HHV-6A in patients with multiple sclerosis

HHV-6 DNAaemia in patients with multiple sclerosis in Kuwait

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia

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Increased detection of serum HHV-6 DNA sequences during multiple sclerosis (MS) exacerbations and correlation with parameters of MS disease progression

Extended observations on the association of HHV-6 and multiple sclerosis

Successful treatment of human herpesvirus-6 encephalitis after bone marrow transplantation

The HHV6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach

Immunohistological analysis of T lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis

Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein

Simultaneous presence of endogenous retrovirus and herpes virus antigens has profound effect on cell-mediated immune responses: implications for multiple sclerosis

Human herpesvirus 6: an emerging pathogen

Microbial triggers of chronic human illness

Detection of antibodies directed against human herpesvirus 6 U94/REP in sera of patients affected by multiple sclerosis

Neuroinvasion and persistence of human herpesvirus 6 in children

Human herpesvirus 6

High frequency of human herpesvirus 6 DNA in multiple sclerosis plaques isolated by laser microdissection

Viruses and multiple sclerosis

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis

Correlation between HHV-6 reactivation and multiple sclerosis disease activity

Association of human endogenous retroviruses with multiple sclerosis and possible interactions with herpes viruses

Human herpesvirus type 6 and multiple sclerosis

HHV-6 and multiple sclerosis

High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay

Update on human herpesvirus 6 biology, clinical features, and therapy

Successful treatment of human herpesvirus 6 encephalomyelitis in immunocompetent patient

Intrathecal antibody (IgG) production against human herpesvirus type 6 occurs in about 20% of multiple sclerosis patients and might be linked to a polyspecific B-cell response

Human herpesvirus 6 (HHV-6) variant B accounts for the majority of symptomatic primary HHV-6 infections in a population of U.S. infants

Multiple sclerosis-associated retrovirus (MSRV) in Sardinian MS patients

Genetics of multiple sclerosis

A genetic basis for familial aggregation in multiple sclerosis: Canadian Collaborative Study Group

Similar humoral and cellular immunological reactivities to human herpesvirus 6 in patients with multiple sclerosis and controls

Autoimmune diseases: genes, bugs and failed regulation

Pathogenesis of multiple sclerosis

HHV-6 and multiple sclerosis

The association of the human herpesvirus-6 and MS

A randomized clinical trial of valacyclovir in multiple sclerosis

Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease

Infectious causes of multiple sclerosis

The search for virus in multiple sclerosis brain

Polymerase chain reaction analysis of human herpesvirus-6 sequences in the sera and cerebrospinal fluid of patients with multiple sclerosis

Human herpesvirus 6 genome and antigen in acute multiple sclerosis lesions

Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity

Activation-induced cell death in T cells

Antibody cross-reactivity between myelin oligodendrocyte glycoprotein and the milk protein butyrophilin in multiple sclerosis

Just another dubious virus in cells from a patient with multiple sclerosis

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity: the Multiple Sclerosis Genetics Group

Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A

Human herpesvirus-6 infection in children: a prospective study of complications and reactivation

Infection of primary human fetal astrocytes by human herpesvirus 6

New concepts in the immunopathogenesis of multiple sclerosis

Absence of Epstein-Barr virus RNA in multiple sclerosis as assessed by in situ hybridisation

Anti-viral properties of interferon beta treatment in patients with multiple sclerosis

A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis

Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial; the IFNB Multiple Sclerosis Study Group

The virology of demyelinating diseases

HHV-6-related secondary graft failure following allogeneic bone marrow transplantation

Human herpesviruses 6 and 7: identification of newly recognized viral pathogens and their association with human disease

Human herpesvirus 6 and multiple sclerosis: systemic active infections in patients with early disease

Epidemiology of multiple sclerosis: does this really point toward an etiology? Lectio doctoralis

Human herpesvirus 6 is closely related to human cytomegalovirus

Temporal relationship between elevation of Epstein-Barr virus antibody titers and initial onset of neurological symptoms in multiple sclerosis

Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis

Epidemiological investigation of the association between infectious mononucleosis and multiple sclerosis

In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6)

Sclerose en plaques et maladies infectieuses

Absence of seven human herpesviruses, including HHV-6, by polymerase chain reaction in CSF and blood from patients with multiple sclerosis and optic neuritis

Human herpesvirus 6 is associated with focal encephalitis

The future of multiple sclerosis therapies: redesigning multiple sclerosis clinical trials in a new therapeutic era

MHC class I-restricted killing of neurons by virus-specific CD8+ T lymphocytes is effected through the Fas/FasL, but not the perforin pathway

Temporal mapping of transcripts in herpesvirus 6 variants

Absence of human herpesvirus 6 and 7 from spinal fluid and serum of multiple sclerosis patients

Association of human herpesvirus 6 with the demyelinative lesions of progressive multifocal leukoencephalopathy

Clonal dominance of CD8+ T-cell in multiple sclerosis

Human herpes virus-6 encephalitis after bone marrow transplantation: successful treatment with ganciclovir

Is Epstein-Barr virus present in the CNS of patients with MS

Immunotherapy for multiple sclerosis: basic insights for new clinical strategies

Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases

Human herpesvirus-6 immunoglobulin G antibodies in patients with multiple sclerosis

Seroepidemiology of human herpesvirus 6 infection in normal children and adults

A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients

Early and late HHV-6 gene transcripts in multiple sclerosis lesions and normal appearing white matter

Antiviral prophylaxis may prevent human herpesvirus-6 reactivation in bone marrow transplant recipients

Human herpesvirus-6 meningoencephalitis in a recipient of an unrelated allogeneic bone marrow transplantation

Fatal primary infection due to human herpesvirus 6 variant A in a renal transplant recipient

Human herpesvirus 6 infects the central nervous system of multiple sclerosis patients in the early stages of the disease

Lymphocyte-mediated cytotoxicity

Cellular localization of human herpesvirus-6 in the brains of children with AIDS encephalopathy

Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders

Detection of herpesviridae in postmortem multiple sclerosis brain tissue and controls by polymerase chain reaction

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Clinical viral infections and multiple sclerosis

Immunosuppressive-associated leukoencephalopathy in organ transplant recipients

Human herpesvirus 6 and multiple sclerosis: survey of anti-HHV-6 antibodies by immunofluorescence analysis and of viral sequences by polymerase chain reaction

Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA

Increased lymphoproliferative response to human herpesvirus type 6A variant in multiple sclerosis patients

Immunology of multiple sclerosis

Multiple sclerosis: a two-stage disease

Myelin-specific CD8 T cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis

Transcriptional analysis of targets in multiple sclerosis

An altered immune response to Epstein-Barr virus in multiple sclerosis: a prospective study

Infectious agents and multiple sclerosis-are Chlamydia pneumoniae and human herpes virus 6 involved?

Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis

Detection of viral DNA and immune responses to the human herpesvirus 6 101-kilodalton virion protein in patients with multiple sclerosis and in controls

Fatal herpesvirus-6 encephalitis in a recipient of a T-cell-depleted peripheral blood stem cell transplant from a 3-loci mismatched related donor

Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allogeneic stem cell transplantation

Association of human herpesvirus 6 and human herpesvirus 7 with demyelinating diseases of the nervous system

Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo

Intrathecal application of interferon gamma: progressive appearance of MHC antigens within the rat nervous system

The immune response against herpesvirus is more prominent in the early stages of MS

Co-localization of human herpes virus 6 and tissue plasminogen activator in multiple sclerosis brain tissue

Human herpesvirus 6 limbic encephalitis after stem cell transplantation

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A potential role for human herpesvirus type 6 in nervous system disease

Heterogeneity of CD4(+) and CD8(+) T cells

Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein

HHV-6 A-or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum

Identification of human herpesvirus-6 as a causal agent for exanthem subitum

Human herpesvirus 6 meningoencephalitis successfully treated with ganciclovir in a patient who underwent allogeneic bone marrow transplantation from an HLA-identical sibling

Central nervous system complications in human herpesvirus-6 infection

MSRV pol sequence copy number as a potential marker of multiple sclerosis

Effect of antivirals on human herpesvirus 6 replication in hematopoietic stem cell transplant recipients