Role of Viral Infections in Multiple Sclerosis Pathogenesis among Indian Population
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.319209
Source of Support: None, Conflict of Interest: None
Keywords: Indian population, multiple sclerosis, pathogenesis, viral infectionKey Message: Certain neurotropic viruses may have a role in triggering relapses through a peripheral mechanism, rather than a direct role through intrathecal multiplication and resultant autoimmune demyelination.
Multiple sclerosis (MS) is a chronic autoimmune inflammatory disorder of the central nervous system affecting predominantly young women worldwide. A recent epidemiological study from coastal Karnataka suggests that the prevalence of MS is likely to be at least 8.3/100,000. In a country of >1.2 billion people, there are approximately100,000 patients with MS, which is nearly 8 times more than what was projected a decade earlier. It is not clear whether risk factors associated with MS in regions of high prevalence are relevant in areas where the disease is less prevalent, as in India. A variety of viruses including measles, human herpesvirus 6 (HHV-6), herpes simplex virus type 1 (HSV-1), and Epstein–Barr virus (EBV) are considered as potential viral agents linked with MS among white populations.
There are limited studies from Indian population. A strong association has been reported with childhood-onset measles among MS patients. An association with mumps in childhood was reported in a small cohort of 63 MS patients and matched controls. There was, however, no association found between MS and remote infection with EBV in Indian patients. There is a significant body of evidence that supports a viral etiology for MS. The candidate viruses associated include herpes group of viruses namely HHV-6,, EBV,, HSV-1,, and human endogenous retrovirus (HERV). A growing body of evidence suggests the involvement of HERV-W, particularly detection of HERV-W and HERV-H molecules in MS patients and increased number of HERW-W DNA copies in genomic DNA from peripheral blood mononuclear cells (PBMC) in secondary progressive MS. HERV-W elements encode a powerful immunopathogenic envelope protein (env) that mediates a pro-inflammatory and autoimmune cascade. Correlation among env gene expression, circulating levels of env protein, and disease activity has been reported. A recently identified MS-associated retrovirus (MSRV) belonging to the HERV-W family produces extracellular virions (MSRV pol gene) in plasma and cerebrospinal fluid (CSF) of MS patients. It is debated whether these associations are a result of the dysregulated immune system consequent to the disease or whether they are causally related to MS. Some studies have correlated new brain lesions with the presence of HHV-6 and EBV., A role as a peripheral trigger in precipitating relapses was supported by a recent study that showed a rising titer of EBV antibody in the peripheral blood associated with new brain lesions.
To our knowledge, a comprehensive study looking at putative mechanisms of viral participation in MS including clinical, radiological, and molecular studies have not been done previously in nonwhite populations where MS prevalence is low. This study is important from the point of understanding what triggers MS in Indians and may have a bearing on the low prevalence of the disease in tropical settings.
Consecutive patients with MS where chosen from the Mangalore demyelinating disease registry. Apart from clinical documentation of disease status (relapse/remission), MRI of the brain was done within 2 weeks of a recent clinical relapse. Presence of new (gadolinium enhancing) lesions was considered as evidence of new lesions. Both blood and CSF samples were obtained and processed with the primary objective of determining whether there was intrathecal multiplication of viruses under study (EBV, HHV6 A and B, and HERV) or a breach in blood–brain barrier (BBB) associated with viral presence in both peripheral blood and CSF. It was expected that presence of viral DNA and reactive oligoclonal bands in the CSF in the presence of an intact BBB would indicate local viral synthesis in relation to MS. A breached BBB that coincided with MS relapse and presence of viral DNA (intrathecal and peripheral blood) would support the role for a virally mediated trigger originating in the periphery that triggered CNS demyelination.
Enrollment of MS patients and controls
A total of 62 MS patients diagnosed by McDonald criteria were included [Table 1] among whom 41 were treatment naive. There were 25 patients with relapsing remitting (RR), 34 with secondary progressive (SP), and 3 with primary progressive (PP) MS. Among patients with MS, 37 patients were in relapse at the time of inclusion in the study and 25 were in remission. Twelve patients who had initially been studied during relapse were also available for the study during the remission phase (≥3 months after last relapse) of illness. Sixty-five controls were included in this study. The controls were patients with neurological illness such as headache, dementia, and epilepsy who underwent lumbar puncture as part of their neurological workup. The study was approved by the institutional ethics committee and patients were enrolled after obtaining informed consent.
Patient samples collection and processing [Figure 1]
CSF: Following a traumatic lumbar puncture, 10 mL of CSF was collected and processed further. Paired samples were used for detection of oligoclonal bands using isoelectric focusing (Hydrasys2, Sebia, France). Patterns 2 and 3 were identified as markers of intrathecal IgG synthesis. Ratio of CSF to blood albumin level was used for testing BBB integrity. CSF/serum albumin index of <9 was considered as normal.
CSF processing for cell lysates and cell-free fractions
CSF was centrifuged immediately (1000 g × 10 min) after collection. Cell-free CSF was stored separately at −80°C. The pellet was resuspended in 50 mL of lysing buffer (1% NP40, 20% Tween, 2.5 mM MgCl2, 10 mM Tris, 100 mM KCl, 120 mg/mL proteinase K), and incubated at 56°C for 1 h, and then treated at 95°C for 15 min. The lysate was stored at −80°C. DNA was extracted from the cell-free CSF as per manufacturer's instructions (NucleoSpin Dx Virus, Macherey-Nagel, Duren, Germany).
Blood sample processing
20 mL of whole blood was collected and processed [Figure 1]. Serum and plasma were separated and stored in aliquots at −80°C for further studies. PBMS were obtained (Ficoll-Paque method). From the PBMCs, cell-associated DNA was extracted using DNA blood kit (NucleoSpin Blood, Macherey-Nagel Duren, Germany) and stored at −80°C. From the cell-free plasma, DNA was extracted using Nucleospin virus kit (Macherey-Nagel, Duren, Germany) and stored at −80°C.
Viral DNA and viral antibody detection for EBV, HHV-6 A and B
DNA extracted from blood and CSF samples (cell-free and cell-associated) were amplified by quantitative PCR (qPCR). The PCR primers (Applied Biosystems, UK) for the Taqman assay were selected from the EBV DNA genome encoding the nonglycosylated membrane protein BNRF1 p143 and HHV6 variants. For standardization of the assay, a standard containing EBV (B 95-8 strain), HHV6A (GS strain), and HHV6B (Z-29 strain) quantified viral DNA with copy number of 2.4 × 104 DNA copies/μL was obtained (Advanced Biotechnologies Inc, Columbia). PCR was done as per protocols, and viral load was estimated.
Viral antibody testing
In the serum, anti-EBV (EBNA1 IgG) and anti-HHV-6 IgG were tested using ELISA kits from Abnova (Taiwan) as per manufacturer's instructions.
RNA extraction from cell-free plasma and CSF sample
200 μL of cell-free samples (plasma and CSF) was filtered through 0.45 μm membranes and treated with bovine ribonuclease 1A (20 μg/mL) and incubated at 37°C for 30 min. RNA was extracted using Illustra RNAspin Mini RNA isolation kit and used for detection of MSRV pol gene.
Patient samples that were positive for oligoclonal band were processed for affinity immunoblotting. Preliminarily serum and CSF IgG was isolated and purified using protein G NAb spin kit (Thermofisher scientific). The following antigens—HHV6A (purified viral lysate, Advanced Biotechnologies Inc, Columbia), HHV6B (My Biosource, San Diego, USA), and EBNA1 (RayBiotechInc, USA) were used as primary antigens, and diluted patient IgG (serum1:250, CSF 1:25) was the source of the primary antibody.
Detection of MSRV pol gene
RNA was converted to cDNA using high capacity RNA to cDNA conversion kit (Thermofisher) as per the instructions. A nested PCR was done for MSRV pol gene. The product of 435 bp was identified by gel electrophoresis [Supplementary Figure 1] and the specificity of the product was confirmed by sequencing (Sigma, Bangalore).
Our study involved 62 MS patients and 65 controls. During a clinical relapse, we evaluated the MRI for evidence of new lesions and increased permeability of the BBB by measuring the CSF/serum albumin index. We looked for evidence of HHV6 A and B, EBNA, and MSRV in the peripheral circulation and intrathecally among both patients and controls in relation to the clinical status of patients (relapse/remission).
Evidence for breach in BBB
MRI evidence of new lesions were seen in 20/37 (54%) patients with clinical relapse. CSF/serum albumin index was abnormal in 32/37 (86.5%) patients.
Viral presence in peripheral blood and CSF
Overall viral antibodies were detected in serum more in patients when compared to controls. EBNA1 IgG was seen in 89% of MS patients and 58% controls (P = <0.001). However, HHV6 IgG was similar in both groups (85% versus 81%; P = 0.45). 11% (7/62) of patients and 11% (7/65) of controls had cell-associated EBV DNA. Cell-free EBV DNA was detected in 8% (5/65) of healthy controls and 5% (3/62) patients. 5% of patients (3/62) with MS had CSF cell-associated and cell-free EBV DNA, but it was absent among control CSF samples. HHV 6B DNA was found in 69% of patients (in both CSF and peripheral blood samples) as compared to 55.4% of plasma samples among controls [Supplementary Table 1]. The difference was not statistically significant (P = 0.10). There was no evidence of HHV6A DNA in cases and controls.
We further attempted to determine whether there was evidence of viral multiplication during relapse. In three patients, there was significant elevation in mean copy number of plasma EBV DNA (remission = 0, relapse = 193) and a trend for viral multiplication during relapse, in cell-associated EBV DNA. In a subset of MS patients, there was a similar increase in viral load during relapse when compared to remission among patients harboring HHV 6B DNA in both CSF and peripheral blood. Mean copy number (viral load) was high for HHV6 during relapse in serum (3477 vs 2245, P = 0.87) as compared to remission phase [Supplementary Table 2].
There was no significant difference in the presence of HERV among patients and controls. MSRV-pol gene amplification revealed that 98% of patients and 94% of controls harbored HERV viral DNA in peripheral blood while the same was true for 87% of patients and 80% of controls in the CSF. There were no patients who had exclusive viral presence or evidence of viral multiplication in CSF alone, for any of the viruses tested in this study.
Results of affinity immunoblotting
Out of the 62 paired samples tested, 33 (53%) patient samples showed oligoclonal band positivity. In affinity immunoblotting reaction, intrathecal IgG synthesis against EBNA1 antigen was demonstrable in 26% (16/62) of patients and none against HHV6 [Figure 2].
MS is a complex disorder in which genetic susceptibility and environmental factors are equally important. Genetic susceptibility for MS is similar among different populations worldwide, but environmental associations may differ geographically. A considerable body of evidence points toward a viral association in MS among white populations though the exact role in MS pathogenesis is unclear. In this study, we evaluated some of the strong viral candidates that have been associated with MS among Europeans namely EBV,, HHV6,, HSV-1,, and HERV [Table 2],,,.
Overall viral association was strong in MS patients as compared to controls and specifically for EBV. EBNA1 IgG prevalence was significantly higher (P = 0.001) in cases (89%) as compared to controls (58%). Evidence of viral multiplication was seen in some patients in both blood and CSF during a relapse. This is in keeping with previous studies which indicate that presence of EBNAI IgG in the periphery is a hallmark of MS in European populations.
An association between a humoral response to EBV and MRI evidence of relapse has been reported earlier.
No patient had intrathecal viral multiplication of either EBV or HHV6 independent of peripheral viral presence. We observed an EBV specific intrathecal oligoclonal IgG production in a quarter of MS patients. Previous studies have suggested that an EBV-specific intrathecal oligoclonal IgG production can occur in MS patients and may result from chronic immune stimulation associated with MS brain inflammation. We found no active role for MSRV infection.
Based on the results of our study, we believe that viruses studied, namely EBV and HHV6, have a role in triggering relapses through a peripheral mechanism, rather than a direct role through intrathecal multiplication and resultant autoimmune demyelination. The role of vaccines in MS is not clearly established. The role in preventing infection, impact on MS disease triggering and potential effect on clinical course and additionally their efficacy when administered with newer disease modifying agents are all areas that need careful consideration. In every patient, the potential prevention of infections that cause MS exacerbation by vaccination has to be balanced against the risks attributed to the same.
There were several limitations to our study including the small number of patients included and that our study was limited to a few neurotropic viruses known to be associated with MS. Despite the limitations, the results of this study in a cohort of Indian patients suggest that viral infections may trigger relapses in MS rather than have a direct effect in disease causation.
This study was funded by the Ministry of Science and Technology, Department of Biotechnology, Government of India. (BT/PR10844/MED/30/1317/2014).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]
[Table 1], [Table 2]