Molecular evidences for a role of HSV-1 in multiple sclerosis clinical acute attack

To verify the possible role of human herpesviruses as triggering or aggravating factors in relapsing-remitting multiple sclerosis (RRMS) clinical acute attack, we studied the prevalence of some herpesviruses in the peripheral blood mononuclear cells (PBMCs) collected from 22 MS patients during an MS relapse and in a stable phase and from 18 healthy controls (HC). DNA belonging to Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), Human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) has been searched by specific nested polymerase chain reaction (n-PCR). EBV and HHV6 DNA has been detected with high frequency in acute and stable MS and in healthy controls without significant differences. HCMV DNA was observed both in acute and stable MS but not in HC, and, more interestingly, HSV-1 DNA was only found in 13% of acute MS, while both stable MS and healthy controls were negative. On the basis of these results we focused on HSV-1, and to confirm them and to demonstrate that HSV-1 is actively replicating in MS patients during clinical relapse, we searched both messenger RNA (mRNA) and DNA of HSV-1 in the PBMCs of 15 acute MS patients and 15 healthy controls. We found HSV-1 mRNA and DNA in a significant number of acute MS patients but not in the control group. On the whole these data indicate that HSV-1 reactivate in the peripheral blood of MS patients during clinical acute attack and probably play a role in the triggering of MS relapses.     

Varicella-zoster virus at relapses of multiple sclerosis

Abstract The possible participation of different herpes viruses was studied during exacerbations of multiple sclerosis (MS). We searched for the presence of DNA from the following herpes viruses: varicella zoster virus (VZV), herpes-simplex viruses 1 and 2; Epstein-Barr virus (EBV) and human herpes-virus-6 (HHV6) in mononuclear cells from patients with MS during relapse (n = 40), MS during remission (n = 131) and controls (n = 125). Additionally, immune cells containing viral antigens were quantified by flow cytometry, and VZV load was determined by real time PCR in 2 MS patients at various times during relapse and remission. DNA from VZV was found in 95% of MS patients during relapse and in 17% during remission; all controls were negative; by contrast, DNA from HHV6 was found in 24% of MS patients during relapse and in 2% during remission; DNA from herpes simplex viruses was not found in any subject; and DNA from EBV was found in a similar percentage of subjects from all groups. Sequential quantification of VZV-load showed a curve that increased during relapse and disappeared at remission. Also, VZV antigens were found inside a large number of immune cells from MS patients during relapse as compared with MS patients on remission and controls. In the typical forms of VZV infection, varicella and herpes-zoster, DNA from VZV is found in mononuclear cells exclusively during brief periods at the beginning of the active infection, but not during latency; thus, the conspicuous presence of VZV during relapses of MS may indicate a period of active infection and suggests the participation of VZV in the pathogenesis of MS.     

多发性硬化患者血清和脑脊液Epstein-Barr病毒抗体检测的意义

目的探讨多发性硬化(MS)患者血清和脑脊液(CSF)中EpsteinBarr(EB)病毒IgG抗体检测的意义。方法采用酶联免疫吸附法检测MS患者65例、其他神经科疾病(OND组)患者71例、非神经科疾病(NND组)患者42例的血清和CSF中EB病毒核抗原、壳抗原和早期抗原的IgG抗体,并进行分析比较。根据血清抗体检测结果的组合,分析各组中病毒初次感染、既往感染和病毒重新激活的情况。结果3组患者血清EB病毒核抗原、壳抗原IgG抗体阳性率均>90%,差异无显著性(均P>0.05)。MS组EB病毒早期抗原IgG抗体阳性率(46.2%)明显高于其他两组(18.3%,9.5%,均P<0.05)。MS组病毒感染重新激活的比率(46.2%)明显高于其他两组(18.3%,9.5%,均P<0.05)。3组CSF病毒抗体阳性率差异无显著性(均P>0.05)。结论MS患者活动性的EB病毒感染较多,EB病毒感染重新激活的比例很高。     

Association between clinical disease activity and Epstein-Barr virus reactivation in MS

OBJECTIVE: To assess the potential significance of Epstein-Barr virus (EBV) reactivation in disease activity in MS patients. METHODS: The prevalence of antibodies against herpes simplex virus type 1 (HSV-1), HSV-2, EBV, and cytomegalovirus was determined in a group of 108 MS patients and in 163 healthy control subjects. Sera were analyzed using combinations of novel assay systems employing highly purified viral and recombinant antigens. In addition, PCR for the detection of EBV DNA was performed in serial samples. RESULTS: In contrast to the control populations, antibodies against EBV were present in 100% of MS patients. Among the tested human herpesviruses, this high extent of seropositivity was only found for EBV. Primary infection was found exclusively in the control group (3.7%), whereas serologic evidence of EBV reactivation was seen in MS patients (13. 9%) as well as control subjects (17.2%). There was no temporal coincidence between EBV reactivation and disease activity in MS patients. However, in 19 patients followed monthly for 1 year, active viral replication as measured by increased immunoglobulin (Ig) M and IgA responses to EBV early antigens (p54 + p138) and positive serum DNA was seen in 72.7% of patients with exacerbations during the study period and in none of the patients with clinically stable disease. CONCLUSIONS: The results demonstrate an association between EBV reactivation and disease activity in MS patients over time, and suggest that EBV might play an indirect role in MS as an activator of the underlying disease process.     

The significance of Epstein—Barr virus seropositivity in multiple sclerosis patients?

The objective of this study was to evaluate and investigate the significance of the previously found 100% seropositivity toward Epstein–Barr virus (EBV) found in multiple sclerosis (MS) patients in contrast to healthy controls. Using a commercially available ELISA-test (Biotest), which differentiates infections with EBV into previous infections, primary infections, reactivated infections and no previous infection, we found 137 of 138 MS patients and 124 of 138 healthy controls seropositive. A primary infection in 4 of the 124 EBV seropositive healthy controls in contrast to no primary infections in the MS EBV seropositive group was significant ( P =0.049652, Fishers exact test). This may be suggestive of a lack of primary infections in MS patients, and thus strengthens the idea that MS patients are infected with EBV before development of MS. Further studies are in progress to analyse whether EBV infection is a prerequisite for the development of this disease.     

Seroreactivity to human T cell leukemia/lymphoma virus type 1 and related retroviruses in multiple sclerosis patients from Denmark and the Faroes

A total of 40 multiple sclerosis (MS) patients from Denmark and 10 from the Faroes were examined for antibodies with affinity to human T cell leukemia/lymphoma virus type 1 (HTLV-I) and human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2). Using ELISA, MS patients and a group of healthy controls did not differ significantly in their reactivities to HTLV-I. However, elevated reactivities were recorded with 5 MS sera, whereas only 2 of the sera from the controls produced highly values. Ten patients with other neurological diseases all seemed to exhibit low reactivity in HTLV-I ELISA. The reactivities of 2 MS sera decreased considerably by absorption with an HTLV-I lysate. In immunofluorescence assay, two other MS sera reacted with HTLV-I transformed cell lines as well as with non-infected cells. Examined by Western blotting (WB), a single MS serum produced a distinct HTLV-I p19 band. With ELISA for detection of HIV-1 and HIV-2 antibodies, 2 MS sera exhibited borderline reactions. Further examination of these two sera by WB revealed weak reactivities against p24 and p53 of HIV-1. One the whole, the present observations do not suggest that a putative MS retrovirus would be closely related with HTLV-I, HIV-1 or HIV-2.     

A role of late Epstein-Barr virus infection in multiple sclerosis

OBJECTIVE: To assess Epstein-Barr virus (EBV) seroconversion in a high multiple sclerosis (MS) prevalence area and to evaluate the recall of diagnosed infectious mononucleosis in MS patients. METHODS: The study was based on information or blood samples, or both, from schoolchildren, young MS patients and matched controls. EBV serology was performed on 1154 blood samples. RESULTS : We demonstrate that more than one third of the population in a high MS prevalence area is seronegative to EBV at puberty. This is in contrast to the virtually complete seroconversion to EBV early in life in individuals from areas with a low prevalence of MS. Furthermore, we demonstrate that recall of diagnosed infectious mononucleosis (IM), but not recall of common childhood diseases, is significantly more frequent among MS patients than healthy controls. All MS patients, including patients without prior immunosuppressive treatment, were EBV seropositive. CONCLUSION: During or after puberty, EBV is transmitted to a major proportion of the population in an MS high-prevalence area. Together with our previous documentation of an association between late infection with EBV and an increased risk of developing MS, these data support a role of EBV infection in MS.     

Epstein-Barr virus genotypes in multiple sclerosis

Background –  Multiple sclerosis (MS) is associated with Epstein–Barr virus (EBV) infection, but the relationship between the virus and the disease is not clear. As many different types of EBV exist, it is possible that MS is caused by one particular type of EBV.
Objectives –  The aim of this study was to determine whether MS is associated with a particular genotype of EBV.
Materials and methods –  We collected blood from MS patients and controls, amplified and sequenced the latent membrane protein-1 (LMP-1) gene, and compared the groups.
Results –  We found a variety of LMP-1 sequences in both MS and controls, with no significant differences between the groups.
Conclusion –  We conclude that MS is not associated with a particular genotype of EBV.     

Presence of Epstein-Barr virus and human herpesvirus 6B DNA in multiple sclerosis patients: associations with disease activity

OBJECTIVE: To assess the presence of Epstein-Barr virus (EBV) and human herpesvirus 6B (HHV-6B) DNA in saliva and plasma from multiple sclerosis (MS) patients enrolled in a randomized, double-blind, placebo-controlled valacyclovir treatment study. METHODS: DNA was prepared following ultracentrifugation of saliva and plasma. EBV and HHV-6B DNAs were determined by real-time polymerase chain reaction. RESULTS: EBV and HHV-6B DNAs were detected in 41% and 65% of saliva samples, respectively. In patients treated with valacyclovir, the percentage of saliva samples with EBV was significantly reduced (9%; P = 0.000017), whereas the frequency of HHV-6B positive samples was unchanged (57%; P = 0.38). Longitudinal studies demonstrated a time-dependent reduction in the frequency of saliva samples containing EBV following valacyclovir treatment. In contrast, plasma contained EBV and HHV-6B DNAs in 17% and 25% of the samples, respectively, and these numbers were not significantly reduced following valacylovir treatment (13% and 16%, respectively), nor were they different from those of healthy controls (6% and 39%, respectively). Patients with high disease activity had a significantly higher frequency of EBV (P = 0.018) and HHV-6B (P = 0.023) positive samples than did patients with low disease activity. The presence of EBV and HHV-6B was strongly correlated in plasma (P < 0.00000001), but not in saliva (P = 0.41). CONCLUSION: MS patients express EBV and HHV-6B in both saliva and plasma, but only the expression of EBV in saliva is significantly reduced following valacyclovir treatment. Although EBV and HHV-6B DNAs can be detected in plasma from healthy individuals, the co-expression of both these viruses in MS patients is highly significant and further associated with clinical activity. The observations of viral DNA in plasma are consistent with an underlying immunologic defect in MS.     

Quantitative PCR for Epstein-Barr virus DNA and RNA in multiple sclerosis

BACKGROUND: Epstein-Barr virus (EBV) is associated with MS, but it is not clear whether EBV plays a role in the pathogenesis of MS. HYPOTHESIS: We hypothesized that the immune control of EBV might be defective in MS, and that reactivation of EBV might drive the immune response in MS. METHODS: We collected blood from controls and patients with MS, and measured the amounts of EBV DNA and RNA using quantitative PCR. RESULTS: We found that EBV DNA and RNA were frequently detectable in peripheral blood leukocytes from both patients with MS and normal controls. There was no significant difference between patients with MS or controls. Paired samples from a small number of subjects suggest that EBV DNA may increase before and during clinical relapse. CONCLUSIONS: We conclude that the immune control of EBV infection is similar in MS and controls, and that reactivation of EBV may correlate with MS disease activity.     

Epstein–Barr virus reactivation and multiple sclerosis

Infection with Epstein–Barr virus (EBV) is considered one of the possible key environmental factors in the aetiology of multiple sclerosis (MS). Whether EBV plays an underlying role as an activator of MS remains, however, controversial. Sixty-one patients with definite relapsing–remitting multiple sclerosis (RRMS) according to the Poser criteria were followed for 1 year. Blood samples were drawn at baseline, months 3, 6 and 12, and in case of any clinical exacerbation. Twenty-three baseline–paired exacerbation samples in the same set were quantitatively analysed to examine whether exacerbations in MS were associated with a change in anti-diffuse component of the EBV-early antigen (EA-D) IgG ratio. All the 61 patients (100%) were anti-viral capsid antigen (VCA) IgG positive, one (2%) was anti-VCA IgM positive and 60 (98%) were anti-EBV nuclear antigen IgG positive. Mean anti-EA-D IgG at baseline was 0.57 (range 0.12–2.70) and at the time of exacerbations 0.61 (range 0.11–2.70). Wilcoxon signed rank test revealed no differences between the 23 baseline and paired exacerbation samples ( P  = 0.58). Our findings suggest that reactivation of latent EBV infection does not play a significant role for exacerbations in RRMS.     

Epstein–Barr virus neutralizing and early antigen antibodies in multiple sclerosis

Background: Our objective was to determine whether antibodies against the Epstein–Barr virus (EBV) nuclear antigen‐1 (EBNA‐1), early antigen (EA), and EBV neutralizing antibodies (NeutAb) are altered in multiple sclerosis (MS). Methods: We measured EBNA‐1 IgG, EA IgG, and EA IgA using quantitative ELISA. We measured NeutAb using a quantitative competitive ELISA. We studied 80 patients with MS, 80 matched controls, and 19 patients with MS with samples collected both whilst stable and in relapse. Results: Epstein–Barr virus nuclear antigen‐1 IgG and EA IgA were increased in MS compared to controls. The EBNA‐1 index value was 23.3 ± 18.3 in the patients with MS (mean ± SD) and 16.3 ± 17.4 in the controls (P = 0.007, paired t‐test). EA IgA had a median value of 1.964 in the patients with MS and 1.248 in the controls (P = 0.029, Wilcoxon signed rank test). EA IgG and NeutAb were not significantly different. None of the antibody levels were altered in relapse. The correlation between concentrations of different antibodies was minimal. Conclusions: IgG antibodies to EBNA‐1 are significantly increased in MS. IgA antibodies against EBV EA are also increased. The EBV neutralizing antibody response is similar in MS and controls.     

Leukotropic herpesviruses in multiple sclerosis

Detection frequency of human herpesvirus 6 (HHV-6) and Epstein Barr virus (EBV) DNA in multiple sclerosis (MS) patients and controls was investigated. DNA of peripheral blood mononuclear leukocytes (PBL) was isolated and amplified by polymerase chain reaction techniques. EBV DNA was detected in all patients and controls. HHV-6 DNA was detected in 7% of MS patients and in 14% of controls. Results are compared with other investigations of HHV-6 DNA in PBL, serum, CSF and brain of patients with MS. Results of the present study and other investigations do not show an association between HHV-6 in PBL and MS. Multiple Sclerosis (2000) 6, 66 - 68     

Epstein-Barr virus shedding by astronauts during space flight

Patterns of Epstein-Barr virus (EBV) reactivation in 32 astronauts and 18 healthy age-matched control subjects were characterized by quantifying EBV shedding. Saliva samples were collected from astronauts before, during, and after 10 space shuttle missions of 5-14 days duration. At one time point or another, EBV was detected in saliva from each of the astronauts. Of 1398 saliva specimens from 32 astronauts, polymerase chain reaction analysis showed that 314 (23%) were positive for EBV DNA. Examination by flight phase showed that 29% of the saliva specimens collected from 28 astronauts before flight were positive for EBV DNA, as were 16% of those collected from 25 astronauts during flight and 16% of those collected after flight from 23 astronauts. The mean number of EBV copies from samples taken during the flights was 417 per mL, significantly greater (p<.05) than the number of viral copies from the preflight (40) and postflight (44) phases. In contrast, the control subjects shed EBV DNA with a frequency of 3.7% and mean number of EBV copies of 40 per mL of saliva. Ten days before flight and on landing day, titers of antibody to EBV viral capsid antigen were significantly (p<.05) greater than baseline levels. On landing day, urinary levels of cortisol and catecholamines were greater than their preflight values. In a limited study (n=5), plasma levels of substance P and other neuropeptides were also greater on landing day. Increases in the number of viral copies and in the amount of EBV-specific antibody were consistent with EBV reactivation before, during, and after space flight.     

Epstein-Barr virus and multiple sclerosis

This review of the considerable evidence linking Epstein-Barr virus (EBV) infection to risk and disease progression in multiple sclerosis (MS) builds on the background to the virus and its interactions with the human host available in the online supplement (see supplement, available online only). The evidence for a similarity in the geographic patterns of occurrence of MS and EBV infection (with infectious mononucleosis or EBV specific serology used as surrogate markers), when reviewed critically, is very limited. There is strong evidence however that people with MS are more likely to report a past history of infectious mononucleosis (thought to represent initial EBV infection at an older age), and higher titres of EBV specific antibodies are associated with an increased risk of developing MS. Elevated levels of the latter are apparent many years before MS onset (compared with non-MS controls) and there is a dose-response relationship between MS risk and antibody titre, with antibodies to the EBV nuclear antigen-1 particularly important. The evidence in relation to EBV DNA load in blood or CSF is conflicting, as is that in relation to T cell responses to EBV. Several hypotheses that have been proposed to explain the links between EBV and MS risk are reviewed and gaps requiring further research are identified.     

The implications of Epstein-Barr virus in multiple' sclerosis - a review

The objective of this article is to bring together knowledge about Epstein-Barr virus (EBV) in relation to multiple sclerosis (MS) in order to evaluate its implications in this disease. All MS patients are EBV seropositive, but EBV is not normally detected in the brain. EBV can explain many of the epidemiological dogmas known in MS. In addition, other studies point towards the involvement of EBV in MS. Despite this, other co-actors seem also to be involved. We still need to know whether EBV may be an initiating factor in MS or whether it is a factor in the pathogenesis. Possible ways of EBV involvement are discussed: direct involvement, an autoimmune inducing factor or a transactivating factor. A current treatment study of MS patients with a specific herpes antiviral drug may add further information to the etiology and pathogenesis of MS.     

High seroprevalence of Epstein-Barr virus in children with multiple sclerosis

We studied seroprevalence and concentrations of Epstein-Barr virus (EBV) antibodies in 147 pediatric patients with multiple sclerosis (MS) and paired controls. The children with MS showed a near-complete seropositivity for EBV antibody against virus capsid antigen (98.6% vs 72.1% in controls, p = 0.001) but did not display serologic evidence for a recent EBV infection. EBV antibody concentrations of pediatric patients with MS were significantly higher vs controls.     

HTLV-I sequences are not detected in peripheral blood genomic DNA or in brain cDNA of multiple sclerosis patients

Human T-cell lymphotropic virus (HTLV-I) was recently reported to be etiologically associated with multiple sclerosis (MS). Genomic DNA from peripheral blood lymphocytes and brain plaques of patients with MS was analyzed for the presence of sequences homologous to the HTLV-I pol gene using the polymerase chain reaction and dot blot techniques. Comparison of DNA amplification patterns between patients with MS, and with control subjects who have other autoimmune conditions, with those in healthy control subjects and with an HTLV-I-infected cell line indicates that HTLV-I pol sequence is not present in the peripheral blood of patients with MS, and that the virus is not active in MS brain plaques.     

Neuropathology of human T lymphotropic virus type I-associated myelopathy

In order to clarify pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) a detailed neuropathological analysis of eight autopsy patients with HAM/TSP was performed. Inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the middle to lower thoracic spinal cords and were extended continuously to the entire spinal cord. Horizontal distribution of inflammatory lesions was symmetric at any spinal levels. Immunohistochemical analysis demonstrated T cell dominance. The numbers of CD4+ T cells and CD8+ T cells were present in equal numbers in patients with shorter clinical course. Apoptosis of helper/inducer T cells were observed in the presence of TIA1+ cytotoxic T cells in these active inflammatory lesions. Inflammatory infiltrates were markedly decreased and CD8+/TIA1- T cells were predominated over CD4+ cells in patients with prolonged clinical course. HTLV-I proviral deoxyribonucleic acid (DNA) amounts in the freshly frozen spinal cord measured by quantitative polymerase chain reaction (PCR) were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV-I provial DNA using multi-primar pairs demonstrated the presence of HTLV-I infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that the target of the inflammatory process seen in HAM/TSP lesions may be HTLV-I infected CD4+ T cells infiltrating the spinal cord.     

Retrovirus-like particles in an Epstein-Barr virus-producing cell line derived from a patient with chronic progressive myelopathy

A B-lymphoblastoid cell line (LCL) of polyclonal origin was isolated from a 30-year-old male patient with a chronic progressive myelopathy clinically resembling multiple sclerosis (MS). The LCL expresses Epstein-Barr virus (EBV) encoded proteins and on transmission electron microscopy (EM) the LCL was shown to produce both EBV particles and retrovirus-like particles spontaneously. The LCL was negative for human retrovirus (HIV-I and HTLV-I) sequences by polymerase chain reaction (PCR). Furthermore the patient was seronegative to these retroviruses including HTLV-II and HIV-II. We, therefore, suggest that the LCL is double-infected with EBV and a hitherto uncharacterized human retrovirus. The possible implications of these two viruses on development of diseases are discussed.