European Journal of Neurology 2014, 21: 1096–1101
doi:10.1111/ene.12435
Identification of the major HHV-6 antigen recognized by cerebrospinal fluid IgG in multiple sclerosis b,c R. Alendaa,b, R. Alvarez-Lafuente , L. Costa-Frossarda,b, R. Arroyob,c, S. Miretea, ~oa,b,d,* and L. M. Villara,b,* J. C. Alvarez-Cerme n a
MS Unit, Departments of Immunology and Neurology, Hospital Ram on y Cajal, IRYCIS, Madrid; bRed Espa~ nola de Esclerosis c M ultiple, Madrid; Department of Neurology, Hospital Clınico San Carlos, IdISSC, Madrid; and dDepartment of Medicine, University of Alcal a, Alcal a de Henares, Madrid, Spain
Keywords:
antibodies, demyelinating diseases, multiple sclerosis, neuroimmunology Received 19 December 2013 Accepted 6 March 2014
Background and purpose: Different data show an association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS). Intrathecal anti-HHV-6 immunoglobulin G (IgG) was detected in MS patients, but the antigen recognized by cerebrospinal fluid (CSF) IgG has not been characterized yet. Our objective was to identify the HHV-6 antigens recognized by IgG present in the CSF of patients with MS. Methods: Cerebrospinal fluid IgG of 15 MS patients and eight patients with other neurological diseases was purified on protein G Sepharose columns. Purified IgG from every patient was linked to a CNBr-activated Sepharose 4B column. Fifty micrograms of viral extract was applied to each column. Bound proteins were eluted and analysed by SDS-PAGE and silver staining. The viral protein was characterized by mass spectrometry. Results: A protein of 150 kD was eluted from CSF IgG columns of three of eight patients with primary progressive MS and one of seven with relapsing remitting MS. After digestion and mass spectrometry analysis 10 peptides were found with 100% homology with the major capsid protein of the HHV-6A. Discussion: These findings confirm the presence of anti-HHV-6 IgG in CSF of MS patients, particularly in progressive forms, and identify major capsid protein as the major antigen recognized by CSF IgG from MS patients.
Introduction Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. Although its aetiology remains unknown, different environmental factors have been associated with MS development. These include vitamin D levels, smoking and infectious agents, such as Epstein Barr virus and human herpesvirus 6 (HHV-6). Different studies have described a relationship between HHV-6 and MS. The presence of DNA and proteins of HHV-6 was found in oligodendrocytes, astrocytes, lymphocytes and microglia of MS plaques [1]. In addition, increased levels of anti-HHV-6-specific antibody levels and HHV-6 DNA were found in Correspondence: L. M. Villar, Servicio de Inmunologıa, Hospital Ram on y Cajal, Carretera de Colmenar Km 9.100, 28034 Madrid, Spain (tel.: +34 913368795; fax: +34 913368809; e-mail: luisamaria. [email protected]). *These authors were co-principal investigators.
1096
the serum of MS patients [2–9], and a correlation was found between the reactivation of HHV-6 and disease activity [9–13]. Moreover, an intrathecal immunoglobulin G (IgG) response to anti-HHV-6 was detected in approximately one-fifth of MS patients [14,15]. This was evidenced by the presence of oligoclonal IgG bands reacting against a total virus extract. However, the HHV-6 antigens recognized by oligoclonal IgG bands present in the cerebrospinal fluid (CSF) of MS patients have not been identified yet. Oligoclonal IgG bands are a hallmark of MS. They demonstrate an antigen driven humoral immune response restricted to the CNS. However, the precise autoantigens recognized by these antibodies remain unknown. In the present study, a method was developed to purify and identify the HHV-6 antigens recognized by IgG present in the CSF of patients with MS and the relationship of these antibodies with different disease forms was studied. This can contribute to identifying autoantigens that play a relevant role in MS © 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
physiopathology and to further demonstrate the relationship of HHV-6 and MS.
Patients and methods Patients and samples
In our study 15 patients diagnosed with MS following the McDonald criteria at the Department of Neurology of the Ram on y Cajal Hospital, Madrid, Spain, were included [16]. Seven showed a relapsing remitting (RRMS) course and eight a primary progressive disease (PPMS). The control group included two patients with normal pressure hydrocephalus, two with stroke, and one with each of the following pathologies: nystagmus, bilateral optic neuritis, Tolosa Hundt syndrome and vasculitis. The study was approved by the Ethics Committee of Ram on y Cajal. CSF samples were collected always for clinical purposes and the remaining CSF was used in our study after obtaining informed consent. All samples were aliquoted and stored at 80 °C until use. Methods
IgG concentration was measured by nephelometry in a Siemens nefelometer BNII. Detection of oligoclonal IgG bands was performed by isoelectrofocusing and western blot as previously described [17].
Cerebrospinal fluid IgG was purified by affinity chromatography protein G Sepharose (GE Healthcare, Little Chalfont, UK) microcolumns (Cytosignal, Irvine, CA, USA). A volume of CSF containing 20 lg of IgG was applied to each column. Bound IgG was eluted with 0.1 M Gly-HCl, pH 2.5, neutralized with 1 M Tris/HCl pH 9.7 (Fig. 1) and dialysed against 0.1 M CO3HNa pH 8.3. Purified IgG was linked to CNBr-activated Sepharose 4B following the manufacturer’s instructions. A column was prepared for every patient. All columns were incubated with 50 lg of viral extracts (HHV-6A GS strain, ABI, Columbia, MD, USA). Bound viral antigens were eluted with 50 mM diethylamine pH 11.5 and analysed by polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Protein bands were characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Peptide identification was performed by a Mascott search. To investigate the presence of potential cross-reacting epitopes in humans from the possible characterized viral proteins, the MHC class I and II predictions from the Immune Epitope Database and Analysis resource (IEDB), a central data repository and service containing MHC binding data relating to B cell and T cell epitopes from infectious pathogens, experimental pathogens and self-antigens (autoantigens), was examined [18,19]. Finally, a BLAST analysis of the amino acid sequences of the predicted epitopes with the genome sequence of Homo sapiens (BLASTP 2.2.28) was performed.
(a)
Figure 1 A scheme of the purification method and identification of viral protein recognized by the IgG present in the CSF of an MS patient. CSF IgG was purified on a protein G Sepharose column. (a) Purified IgG from an MS patient and a control separated by SDSPAGE and silver stained. Purified IgG was linked to CNBr-activated Sepharose 4B. A column was prepared for every patient. Fifty micrograms of viral extract was applied to each column. (b) The viral extract analysed by SDS-PAGE and silver staining. Bound proteins were eluted and analysed by SDS-PAGE and silver staining. (c) A 150 kD protein was identified in three patients with PPMS and a patient with RRMS.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1097
(b)
(c)
1098
R. Alenda et al.
Results To identify the major HHV-6 antigens recognized by IgG antibodies present in the CSF of MS patients, CSF IgG from seven patients with RRMS, eight with PPMS and eight patients with other neurological diseases (control group) was purified. Clinical, laboratory and epidemiological data of the patients are shown in Table 1. Thus 20 lg of CSF IgG from every patient was purified by affinity chromatography and bound to a Sepharose 4B column. A scheme of the purification method is shown in Fig. 1. Then, every column was incubated with 50 lg of HHV-6 and washed with 10 mM Tris/HCl, 0.5 M NaCl, 1 mM EDTA, 0,2% NP40 pH 8.0. After a final wash with 10 mM Tris/ HCl, 1 mM EDTA, pH 8.0, bound antigens were eluted with 50 mM diethylamine pH 11.5 and neutralized with 1 M phosphate pH 6.7. After equilibrating the columns again, the same process was performed with 50 lg of HSV-1 (negative control). Identification of viral proteins eluted from IgG columns of MS patients and controls was performed by SDS-PAGE and silver staining (Fig. 1). No band was detected in eluates obtained after incubating the columns with HSV-1. However, a major band of about
150 kD was detected in the eluates from HHV-6 in IgG columns of three patients with PPMS and one with RRMS (Fig. 2). The 150 kD HHV-6 protein was further characterized by peptide fragmentation and mass spectrometry (MALDI-TOF-MS) and 10 sequences with 100% homology with the major capsid protein (MCP) of the HHV-6A were identified (Fig. 3). The results of homology analysis using BLAST search of deduced amino acid sequences are shown in Table 2 [only those with an expected (E) value < 0.1 were included]. As the genome of HHV-6A exhibits homologies to other viruses, the possible homologies of the predicted epitopes of Table 2 for HHV-6A-MCP with other human herpesviruses were analysed. As can be seen in Table 3, when a BLAST search was performed, E values < 0.1 were found for HHV-6B in 14/15 predicted epitopes of HHV-6A-MCP, showing a high homology; however, when these sequences from HHV-6B were compared with the sequences of the genes shown in Table 2, E values < 0.1 were only found in 11/15 predicted epitopes (those with lower E values in comparison with HHV-6A-MCP). Similarly, E values < 0.1 for HHV-7, another species of the genus Roseolovirus like HHV-6A and HHV-6B, were found in 5/15
Patients
Disease
IgG OCB
Age (years)
Sex
Disease duration (months)
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Control 1 Control 2 Control 3 Control 4 Control 5 Control 6 Control 7 Control 8
RRMS RRMS RRMS RRMS RRMS RRMS RRMS PPMS PPMS PPMS PPMS PPMS PPMS PPMS PPMS Bilateral ON NP hydrocephalus Pseudotumor cerebri NP hydrocephalus Stroke Stroke Tolosa Hundt syndrome Myelitis
Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative
30 18 45 37 28 36 36 58 44 40 44 51 42 37 34 47 70 48 75 62 71 53 42
Female Female Female Female Female Male Male Female Female Male Male Female Female Male Male Female Male Female Male Male Female Male Male
0.1 1 0.1 16 66 95 15 84 183 55 74 60 13 42 71 NA NA NA NA NA NA NA NA
EDSS score 1.5 1 4 1 1.5 1.5 1.5 5.5 5.5 3 6 5.5 6 3.5 6 NA NA NA NA NA NA NA NA
Table 1 Clinical, demographic and laboratory data
Bilateral ON, bilateral optic neuritis; NP hydrocephalus, normal pressure hydrocephalus; PPMS, primary progressive MS; RRMS, relapsing remitting MS; OCB, oligoclonal bands; EDSS, expanded disability status scale.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
Figure 2 IgG antibodies were found against HHV-6 antigens in three of eight PPMS patients, one of seven RRMS patients and in none of the eight patients of the control group.
Figure 3 Highlighted in bold are the 10 sequences of the major capsid protein of the herpesvirus 6A identified in the eluate from CSF IgG columns of MS patients. They show 100% homology with this protein.
predicted epitopes of HHV-6A-MCP; but, comparing the corresponding sequences of HHV-7 with the sequences of the genes shown in Table 2, E values < 0.1 were only found in one predicted epitope: KLNDTMENNLPTSV, the one with the lowest E value in comparison with HHV-6A-MCP. No significant results were found for any of the other human herpesviruses.
Discussion Multiple sclerosis is a heterogeneous disease that develops as an interplay between the immune system and environmental stimuli in genetically susceptible individuals [20]. Increasing evidence indicates that viruses may play a role in MS pathogenesis acting as environmental triggers, Epstein Barr virus and HHV© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1099
6, members of the Herpesviridae family, being amongst the strongest candidates. The strong evidence associating HHV-6 to MS is the presence of HHV-6 DNA or protein in MS demyelinating plaques [21]. Another finding that showed the relationship between HHV-6 and MS was the presence of antiHHV-6 IgG in the CSF of MS patients [14,15,22] However, the viral antigens recognized by these antibodies have not been identified yet. This was explored in 15 MS patients (seven with RRMS and eight with PPMS) and in a control group of eight patients with other neurological diseases. CSF IgG was purified and bound to a column and affinity chromatography was performed using a total viral protein extract. IgG from patients of the control group did not recognize any HHV-6 protein. Conversely, reactivity against HHV-6 in four (26.67%) MS patients was detected, consistent with previously published data [23]. These data further confirm an association between HHV-6 and MS. The target of oligoclonal IgG was identified by mass spectrometry as the MCP of HHV-6. This is a structural protein of the capsomers with a molecular size of 152 kD, necessary to effectively assemble the capside [24]. Different potential mechanisms have been proposed for HHV-6 in MS [23] and include direct toxic actions of virus on infected cells; incorporation of host proteins into virus particles, which triggers antibody production directed against the host proteins; and molecular mimicry. The finding of anti-HHV-6AMCP IgG antibodies in the CSF of some MS patients could be related with this last mechanism. The predicted epitopes of this molecule were investigated and some proteins showing homologue amino acid sequences with a low E value on the genome sequence of H. sapiens were found (Table 2). Some of them could be good candidates for crossreacting mechanisms. An interesting one could be galanin receptor. This molecule is only expressed in the endocrine and nervous system and mediates the effect of galanin, a neuropeptide showing neuroprotective activity [25]. Furthermore, it could be speculated that the presence of single nucleotide polymorphisms in some of these genes could increase the homology of the amino acid sequences with the predicted epitopes from HHV-6A-MCP, resulting in a lower E value and increasing the likelihood of a cross-reaction between the anti-HHV-6A-MCP IgG antibodies and some of the host genes in MS patients. The clinical characteristics of patients showing antiMCP antibodies were explored and it was observed
1100
R. Alenda et al.
Table 2 Genes with amino acid sequences that could cross-react with the predicted epitopes for HHV-6A-MCP (E value < 0.1), encoding proteins with extracellular domains MHC classa
Peptide
Percentile rankb
I I I I
FTQHLSFVDMGL LLSIMTLAAM NLFSAFPIHAFT IMIGNIPLPLAPV
3.20 2.05 1.30 0.20
I
IVQRIDFADILPSV
2.40
I
QLCGEPLV
3.90
I
VISEYCMKSNSCL
3.60
I I
SLTSGSYPELAYV KLNDTMENNLPTSV
0.30 0.40
I I I
GQQAICEVILTPV CLLYSSRAATSII DMMESVTESI
3.20 4.30 1.75
I
DMMESVTESI
1.75
I
RIDFADILPSV
3.10
II
SLQLTFFFPLGIYIP
2.58
II
SLQLTFFFPLGIYIP
2.58
II
TTLRRYYQPIPFHRF
3.18
Sequence length (aa)
E value
protein protein protein protein
311 349 151 1013
0.003 0.024 0.034 0.039
Single-pass membrane protein
341
0.046
Integral to plasma membrane
131
0.054
Multi-pass membrane protein
433
0.057
Plasma membrane Partially membrane-associated
135 1129
0.060 0.064
Plasma membrane Multi-pass membrane protein Single-pass membrane protein
399 429 483
0.069 0.069 0.082
Multi-pass membrane protein
664
0.083
Multi-pass membrane protein
1212
0.089
Single-pass type I membrane protein Multi-pass membrane protein
1152
0.018
346
0.046
195
0.060
Protein (gene)
Subcellular location
Olfactory receptor 1N1 (OR1N1) Galanin receptor type 1 (GALR1) Placenta specific protein 2 (PLAC2) Sodium/potassium-transporting ATPase subunit alpha (ATP1A) Major histocompatibility complex class I-related gene protein (MR1) T-cell receptor alpha chain V region (TCRA) Probable G-protein coupled receptor 22 (GPR22) T-cell receptor beta chain (TRB) Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 (ASAP1) LanC-like protein 1 (LANCL1) Integral membrane protein GPR137C Coiled-coil domain-containing protein 47 (CCDC47) Cyclic nucleotide-gated olfactory channel (CNGA2) Solute carrier family 12 member 2 (SLC12A2) Integrin alpha-M (ITGAM)
Multi-pass Multi-pass Multi-pass Multi-pass
Hydroxycarboxylic acid receptor 1 (HCAR1) Interferon omega-1 (IFNW1)
membrane membrane membrane membrane
Secreted
a
MHC-I and MHC-II binding prediction results; bin the prediction method of the IEDB, percentile ranks go from 0 to 100. Since low percentile ranks are good binders, only those with a percentile rank < 5 were included in the BLAST analysis.
Table 3 Analysis of the possible homology of the predicted epitopes in Table 2 for HHV-6A-MCP with other human herpesviruses (only with E values < 0.1) Peptide
HHV-1
HHV-2
HHV-3
HHV-4
HHV-5
HHV-6B
HHV-7
HHV-8
FTQHLSFVDMGL LLSIMTLAAM NLFSAFPIHAFT IMIGNIPLPLAPV IVQRIDFADILPSV QLCGEPLV VISEYCMKSNSCL SLTSGSYPELAYV KLNDTMENNLPTSV GQQAICEVILTPV CLLYSSRAATSII DMMESV TESI RIDFADILPSV SLQLTFFFPLGIYIP TTLRRYYQPIPFHRF
– – – – – – – – – – – – – – –
– – – – – – – – – – – – – – –
– – – – – – – – – – – – – – –
– – – – – – – – – 0.021 – – – – –
– – – – – – – – – 0.040 – – – – –
0.0003 0.08 0.0006 0.0005 0.00005 – 0.00009 0.0003 0.000005 0.00009 0.0005 0.02 0.01 0.0000002 0.00000002
– – – 0.021 – – – – 0.00003 0.009 0.029 – – – 0.025
– – – – – – – – – – – – – – –
that they were present in three PPMS patients (37% of this group) but only in one (14%) with the RRMS form. Although these preliminary results should be confirmed in a larger series, they suggest a closer
association of HHV-6 and the PPMS form. In this line, a recent study reported the presence of DNA HHV-6 in the brain of MS patients, and particularly in progressive forms [26]. © 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
In summary, this work identifies MCP as the major antigen recognized specifically by IgG purified from the CSF of MS patients. Future studies will show if these antibodies play a role in the pathogenesis of the disease.
Acknowledgements This work was supported by grants PI12/02349 and PI12/00239 from the Instituto de Salud Carlos III (Spain) and grant SAF2012-34670 from the Ministerio de Economıa y Competitividad (Spain). Roberto Alvarez-Lafuente is the recipient of a research contract of the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (Feder) (CP07/00273). Raquel Alenda is the recipient of a research contract of the Red Espa~ nola de Esclerosis M ultiple (REEM) from the Instituto de Salud Carlos III (Spain).
Disclosure of conflicts of interest The authors declare no financial or other conflicts of interest.
References 1. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995; 92: 7440–7444. 2. Merelli E, Bedin R, Sola P, et al. Human herpes virus 6 and human herpes virus 8 DNA sequences in brains of multiple sclerosis patients, normal adults and children. J Neurol 1997; 244: 450–454. 3. Mayne M, Krishnan J, Metz L, et al. Infrequent detection of human herpesvirus 6 DNA in peripheral blood mononuclear cells from multiple sclerosis patients. Ann Neurol 1998; 44: 391–394. 4. Cuomo L, Trivedi P, Cardillo MR, et al. Human herpesvirus 6 infection in neoplastic and normal brain tissue. J Med Virol 2001; 63: 45–51. 5. Friedman JE, Lyons MJ, Cu G, et al. The association of the human herpesvirus-6 and MS. Mult Scler 1999; 5: 355–362. 6. Xu Y, Linde A, Fredrikson S, Dahl H, Winberg G. HHV-6 A- or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum. J Med Virol 2002; 66: 394–399. 7. Tejada-Simon MV, Zang YC, Hong J, Rivera VM, Killian JM, Zhang JZ. Detection of viral DNA and immune responses to the human herpesvirus 6 101kilodalton virion protein in patients with multiple sclerosis and in controls. J Virol 2002; 76: 6147–6154. 8. Al-Shammari S, Nelson RF, Voevodin A. HHV-6 DNAaemia in patients with multiple sclerosis in Kuwait. Acta Neurol Scand 2003; 107: 122–124. 9. Chapenko S, Millers A, Nora Z, Logina I, Kukaine R, Murovska M. Correlation between HHV-6 reactivation and multiple sclerosis disease activity. J Med Virol 2003; 69: 111–117.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1101
10. Garcia-Montojo M, Martinez A, De Las Heras V, et al. Herpesvirus active replication in multiple sclerosis. A genetic control? J Neurol Sci 2011; 311: 98–102. 11. Garcia-Montojo M, De Las Heras V, Dominguez-Mozo M, et al. Human herpesvirus 6 and effectiveness of interferon beta 1b in multiple sclerosis patients. Eur J Neurol 2011; 18: 1027–1035. 12. Berti R, Brennan MB, Soldan SS, et al. Increased detection of serum HHV-6 DNA sequences during multiple sclerosis (MS) exacerbations and correlation with parameters of MS disease progression. J Neurovirol 2002; 8: 250–256. 13. Simpson S Jr, Taylor B, Dwyer DE. Anti-HHV-6 IgG titer significantly predicts subsequent relapse risk in multiple sclerosis. Mult Scler 2012; 18: 799–806. 14. Derfuss T, Hohlfeld R, Meinl E. 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. J Neurol 2005; 252: 968–971. 15. Virtanen JO, F€ arkkil€ a M, Multanen J, et al. Evidence for human herpesvirus 6 variant A antibodies in multiple sclerosis: diagnostic and therapeutic implications. J. Neurovirol 2007; 13: 347–352. 16. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis. Ann. Neurol 2001; 50: 121–127. 17. S adaba MC, Gonz alez Porque P, Masjuan J, AlvarezCerme~ no JC, Bootello A, Villar LM. An ultrasensitive method for the detection of oligoclonal IgG bands. J Immunol Methods 2004; 284: 141–145. 18. Peters B, Sidney J, Bourne P, et al. The design and implementation of the immune epitope database and analysis resource. Immunogenetics 2005; 57: 326–336. 19. Peters B, Sidney J, Bourne P, et al. The immune epitope database and analysis resource: from vision to blueprint. PLoS Biol 2005; 3: e91. 20. Lassmann H, Bruck W, Lucchinetti CF. The immunopathology of multiple sclerosis: an overview. Brain Pathol 2007; 17: 210–218. 21. Virtanen J, Wohler J, Fenton K, Reich DS, Jacobson S. Oligoclonal bands in multiple sclerosis reactive against two herpesviruses and association with magnetic resonance imaging findings. Mult Scler 2014; 20: 27–34. 22. Virtanen JO, Pietil€ ainen-Nicklen J, Uotila L, F€ arkkil€ a M, Vaheri A, Koskiniemi M. Intrathecal human herpesvirus 6 antibodies in multiple sclerosis and other demyelinating diseases presenting as oligoclonal bands in cerebrospinal fluid. J Neuroimmunol 2011; 237: 93–97. 23. Fotheringham J, Jacobson J. Human herpesvirus 6 and multiple sclerosis: potential mechanisms for virusinduced disease. Herpes 2005; 12: 4–9. 24. Littler E, Lawrence G, Liu MY, Barrell BG, Arrand JR. Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6. J Virol 1990; 64: 714–722. 25. Mitsukawa K, Lu X, Bartfai T. Galanin, galanin receptors and drug targets. Cell Mol Life Sci 2008; 5: 1796– 1805. 26. Mancuso R, Hernis A, Cavarretta R, et al. Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis. J Med Virol 2010; 82: 1051–1057.
doi:10.1111/ene.12435
Identification of the major HHV-6 antigen recognized by cerebrospinal fluid IgG in multiple sclerosis b,c R. Alendaa,b, R. Alvarez-Lafuente , L. Costa-Frossarda,b, R. Arroyob,c, S. Miretea, ~oa,b,d,* and L. M. Villara,b,* J. C. Alvarez-Cerme n a
MS Unit, Departments of Immunology and Neurology, Hospital Ram on y Cajal, IRYCIS, Madrid; bRed Espa~ nola de Esclerosis c M ultiple, Madrid; Department of Neurology, Hospital Clınico San Carlos, IdISSC, Madrid; and dDepartment of Medicine, University of Alcal a, Alcal a de Henares, Madrid, Spain
Keywords:
antibodies, demyelinating diseases, multiple sclerosis, neuroimmunology Received 19 December 2013 Accepted 6 March 2014
Background and purpose: Different data show an association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS). Intrathecal anti-HHV-6 immunoglobulin G (IgG) was detected in MS patients, but the antigen recognized by cerebrospinal fluid (CSF) IgG has not been characterized yet. Our objective was to identify the HHV-6 antigens recognized by IgG present in the CSF of patients with MS. Methods: Cerebrospinal fluid IgG of 15 MS patients and eight patients with other neurological diseases was purified on protein G Sepharose columns. Purified IgG from every patient was linked to a CNBr-activated Sepharose 4B column. Fifty micrograms of viral extract was applied to each column. Bound proteins were eluted and analysed by SDS-PAGE and silver staining. The viral protein was characterized by mass spectrometry. Results: A protein of 150 kD was eluted from CSF IgG columns of three of eight patients with primary progressive MS and one of seven with relapsing remitting MS. After digestion and mass spectrometry analysis 10 peptides were found with 100% homology with the major capsid protein of the HHV-6A. Discussion: These findings confirm the presence of anti-HHV-6 IgG in CSF of MS patients, particularly in progressive forms, and identify major capsid protein as the major antigen recognized by CSF IgG from MS patients.
Introduction Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. Although its aetiology remains unknown, different environmental factors have been associated with MS development. These include vitamin D levels, smoking and infectious agents, such as Epstein Barr virus and human herpesvirus 6 (HHV-6). Different studies have described a relationship between HHV-6 and MS. The presence of DNA and proteins of HHV-6 was found in oligodendrocytes, astrocytes, lymphocytes and microglia of MS plaques [1]. In addition, increased levels of anti-HHV-6-specific antibody levels and HHV-6 DNA were found in Correspondence: L. M. Villar, Servicio de Inmunologıa, Hospital Ram on y Cajal, Carretera de Colmenar Km 9.100, 28034 Madrid, Spain (tel.: +34 913368795; fax: +34 913368809; e-mail: luisamaria. [email protected]). *These authors were co-principal investigators.
1096
the serum of MS patients [2–9], and a correlation was found between the reactivation of HHV-6 and disease activity [9–13]. Moreover, an intrathecal immunoglobulin G (IgG) response to anti-HHV-6 was detected in approximately one-fifth of MS patients [14,15]. This was evidenced by the presence of oligoclonal IgG bands reacting against a total virus extract. However, the HHV-6 antigens recognized by oligoclonal IgG bands present in the cerebrospinal fluid (CSF) of MS patients have not been identified yet. Oligoclonal IgG bands are a hallmark of MS. They demonstrate an antigen driven humoral immune response restricted to the CNS. However, the precise autoantigens recognized by these antibodies remain unknown. In the present study, a method was developed to purify and identify the HHV-6 antigens recognized by IgG present in the CSF of patients with MS and the relationship of these antibodies with different disease forms was studied. This can contribute to identifying autoantigens that play a relevant role in MS © 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
physiopathology and to further demonstrate the relationship of HHV-6 and MS.
Patients and methods Patients and samples
In our study 15 patients diagnosed with MS following the McDonald criteria at the Department of Neurology of the Ram on y Cajal Hospital, Madrid, Spain, were included [16]. Seven showed a relapsing remitting (RRMS) course and eight a primary progressive disease (PPMS). The control group included two patients with normal pressure hydrocephalus, two with stroke, and one with each of the following pathologies: nystagmus, bilateral optic neuritis, Tolosa Hundt syndrome and vasculitis. The study was approved by the Ethics Committee of Ram on y Cajal. CSF samples were collected always for clinical purposes and the remaining CSF was used in our study after obtaining informed consent. All samples were aliquoted and stored at 80 °C until use. Methods
IgG concentration was measured by nephelometry in a Siemens nefelometer BNII. Detection of oligoclonal IgG bands was performed by isoelectrofocusing and western blot as previously described [17].
Cerebrospinal fluid IgG was purified by affinity chromatography protein G Sepharose (GE Healthcare, Little Chalfont, UK) microcolumns (Cytosignal, Irvine, CA, USA). A volume of CSF containing 20 lg of IgG was applied to each column. Bound IgG was eluted with 0.1 M Gly-HCl, pH 2.5, neutralized with 1 M Tris/HCl pH 9.7 (Fig. 1) and dialysed against 0.1 M CO3HNa pH 8.3. Purified IgG was linked to CNBr-activated Sepharose 4B following the manufacturer’s instructions. A column was prepared for every patient. All columns were incubated with 50 lg of viral extracts (HHV-6A GS strain, ABI, Columbia, MD, USA). Bound viral antigens were eluted with 50 mM diethylamine pH 11.5 and analysed by polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Protein bands were characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Peptide identification was performed by a Mascott search. To investigate the presence of potential cross-reacting epitopes in humans from the possible characterized viral proteins, the MHC class I and II predictions from the Immune Epitope Database and Analysis resource (IEDB), a central data repository and service containing MHC binding data relating to B cell and T cell epitopes from infectious pathogens, experimental pathogens and self-antigens (autoantigens), was examined [18,19]. Finally, a BLAST analysis of the amino acid sequences of the predicted epitopes with the genome sequence of Homo sapiens (BLASTP 2.2.28) was performed.
(a)
Figure 1 A scheme of the purification method and identification of viral protein recognized by the IgG present in the CSF of an MS patient. CSF IgG was purified on a protein G Sepharose column. (a) Purified IgG from an MS patient and a control separated by SDSPAGE and silver stained. Purified IgG was linked to CNBr-activated Sepharose 4B. A column was prepared for every patient. Fifty micrograms of viral extract was applied to each column. (b) The viral extract analysed by SDS-PAGE and silver staining. Bound proteins were eluted and analysed by SDS-PAGE and silver staining. (c) A 150 kD protein was identified in three patients with PPMS and a patient with RRMS.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1097
(b)
(c)
1098
R. Alenda et al.
Results To identify the major HHV-6 antigens recognized by IgG antibodies present in the CSF of MS patients, CSF IgG from seven patients with RRMS, eight with PPMS and eight patients with other neurological diseases (control group) was purified. Clinical, laboratory and epidemiological data of the patients are shown in Table 1. Thus 20 lg of CSF IgG from every patient was purified by affinity chromatography and bound to a Sepharose 4B column. A scheme of the purification method is shown in Fig. 1. Then, every column was incubated with 50 lg of HHV-6 and washed with 10 mM Tris/HCl, 0.5 M NaCl, 1 mM EDTA, 0,2% NP40 pH 8.0. After a final wash with 10 mM Tris/ HCl, 1 mM EDTA, pH 8.0, bound antigens were eluted with 50 mM diethylamine pH 11.5 and neutralized with 1 M phosphate pH 6.7. After equilibrating the columns again, the same process was performed with 50 lg of HSV-1 (negative control). Identification of viral proteins eluted from IgG columns of MS patients and controls was performed by SDS-PAGE and silver staining (Fig. 1). No band was detected in eluates obtained after incubating the columns with HSV-1. However, a major band of about
150 kD was detected in the eluates from HHV-6 in IgG columns of three patients with PPMS and one with RRMS (Fig. 2). The 150 kD HHV-6 protein was further characterized by peptide fragmentation and mass spectrometry (MALDI-TOF-MS) and 10 sequences with 100% homology with the major capsid protein (MCP) of the HHV-6A were identified (Fig. 3). The results of homology analysis using BLAST search of deduced amino acid sequences are shown in Table 2 [only those with an expected (E) value < 0.1 were included]. As the genome of HHV-6A exhibits homologies to other viruses, the possible homologies of the predicted epitopes of Table 2 for HHV-6A-MCP with other human herpesviruses were analysed. As can be seen in Table 3, when a BLAST search was performed, E values < 0.1 were found for HHV-6B in 14/15 predicted epitopes of HHV-6A-MCP, showing a high homology; however, when these sequences from HHV-6B were compared with the sequences of the genes shown in Table 2, E values < 0.1 were only found in 11/15 predicted epitopes (those with lower E values in comparison with HHV-6A-MCP). Similarly, E values < 0.1 for HHV-7, another species of the genus Roseolovirus like HHV-6A and HHV-6B, were found in 5/15
Patients
Disease
IgG OCB
Age (years)
Sex
Disease duration (months)
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Control 1 Control 2 Control 3 Control 4 Control 5 Control 6 Control 7 Control 8
RRMS RRMS RRMS RRMS RRMS RRMS RRMS PPMS PPMS PPMS PPMS PPMS PPMS PPMS PPMS Bilateral ON NP hydrocephalus Pseudotumor cerebri NP hydrocephalus Stroke Stroke Tolosa Hundt syndrome Myelitis
Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative
30 18 45 37 28 36 36 58 44 40 44 51 42 37 34 47 70 48 75 62 71 53 42
Female Female Female Female Female Male Male Female Female Male Male Female Female Male Male Female Male Female Male Male Female Male Male
0.1 1 0.1 16 66 95 15 84 183 55 74 60 13 42 71 NA NA NA NA NA NA NA NA
EDSS score 1.5 1 4 1 1.5 1.5 1.5 5.5 5.5 3 6 5.5 6 3.5 6 NA NA NA NA NA NA NA NA
Table 1 Clinical, demographic and laboratory data
Bilateral ON, bilateral optic neuritis; NP hydrocephalus, normal pressure hydrocephalus; PPMS, primary progressive MS; RRMS, relapsing remitting MS; OCB, oligoclonal bands; EDSS, expanded disability status scale.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
Figure 2 IgG antibodies were found against HHV-6 antigens in three of eight PPMS patients, one of seven RRMS patients and in none of the eight patients of the control group.
Figure 3 Highlighted in bold are the 10 sequences of the major capsid protein of the herpesvirus 6A identified in the eluate from CSF IgG columns of MS patients. They show 100% homology with this protein.
predicted epitopes of HHV-6A-MCP; but, comparing the corresponding sequences of HHV-7 with the sequences of the genes shown in Table 2, E values < 0.1 were only found in one predicted epitope: KLNDTMENNLPTSV, the one with the lowest E value in comparison with HHV-6A-MCP. No significant results were found for any of the other human herpesviruses.
Discussion Multiple sclerosis is a heterogeneous disease that develops as an interplay between the immune system and environmental stimuli in genetically susceptible individuals [20]. Increasing evidence indicates that viruses may play a role in MS pathogenesis acting as environmental triggers, Epstein Barr virus and HHV© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1099
6, members of the Herpesviridae family, being amongst the strongest candidates. The strong evidence associating HHV-6 to MS is the presence of HHV-6 DNA or protein in MS demyelinating plaques [21]. Another finding that showed the relationship between HHV-6 and MS was the presence of antiHHV-6 IgG in the CSF of MS patients [14,15,22] However, the viral antigens recognized by these antibodies have not been identified yet. This was explored in 15 MS patients (seven with RRMS and eight with PPMS) and in a control group of eight patients with other neurological diseases. CSF IgG was purified and bound to a column and affinity chromatography was performed using a total viral protein extract. IgG from patients of the control group did not recognize any HHV-6 protein. Conversely, reactivity against HHV-6 in four (26.67%) MS patients was detected, consistent with previously published data [23]. These data further confirm an association between HHV-6 and MS. The target of oligoclonal IgG was identified by mass spectrometry as the MCP of HHV-6. This is a structural protein of the capsomers with a molecular size of 152 kD, necessary to effectively assemble the capside [24]. Different potential mechanisms have been proposed for HHV-6 in MS [23] and include direct toxic actions of virus on infected cells; incorporation of host proteins into virus particles, which triggers antibody production directed against the host proteins; and molecular mimicry. The finding of anti-HHV-6AMCP IgG antibodies in the CSF of some MS patients could be related with this last mechanism. The predicted epitopes of this molecule were investigated and some proteins showing homologue amino acid sequences with a low E value on the genome sequence of H. sapiens were found (Table 2). Some of them could be good candidates for crossreacting mechanisms. An interesting one could be galanin receptor. This molecule is only expressed in the endocrine and nervous system and mediates the effect of galanin, a neuropeptide showing neuroprotective activity [25]. Furthermore, it could be speculated that the presence of single nucleotide polymorphisms in some of these genes could increase the homology of the amino acid sequences with the predicted epitopes from HHV-6A-MCP, resulting in a lower E value and increasing the likelihood of a cross-reaction between the anti-HHV-6A-MCP IgG antibodies and some of the host genes in MS patients. The clinical characteristics of patients showing antiMCP antibodies were explored and it was observed
1100
R. Alenda et al.
Table 2 Genes with amino acid sequences that could cross-react with the predicted epitopes for HHV-6A-MCP (E value < 0.1), encoding proteins with extracellular domains MHC classa
Peptide
Percentile rankb
I I I I
FTQHLSFVDMGL LLSIMTLAAM NLFSAFPIHAFT IMIGNIPLPLAPV
3.20 2.05 1.30 0.20
I
IVQRIDFADILPSV
2.40
I
QLCGEPLV
3.90
I
VISEYCMKSNSCL
3.60
I I
SLTSGSYPELAYV KLNDTMENNLPTSV
0.30 0.40
I I I
GQQAICEVILTPV CLLYSSRAATSII DMMESVTESI
3.20 4.30 1.75
I
DMMESVTESI
1.75
I
RIDFADILPSV
3.10
II
SLQLTFFFPLGIYIP
2.58
II
SLQLTFFFPLGIYIP
2.58
II
TTLRRYYQPIPFHRF
3.18
Sequence length (aa)
E value
protein protein protein protein
311 349 151 1013
0.003 0.024 0.034 0.039
Single-pass membrane protein
341
0.046
Integral to plasma membrane
131
0.054
Multi-pass membrane protein
433
0.057
Plasma membrane Partially membrane-associated
135 1129
0.060 0.064
Plasma membrane Multi-pass membrane protein Single-pass membrane protein
399 429 483
0.069 0.069 0.082
Multi-pass membrane protein
664
0.083
Multi-pass membrane protein
1212
0.089
Single-pass type I membrane protein Multi-pass membrane protein
1152
0.018
346
0.046
195
0.060
Protein (gene)
Subcellular location
Olfactory receptor 1N1 (OR1N1) Galanin receptor type 1 (GALR1) Placenta specific protein 2 (PLAC2) Sodium/potassium-transporting ATPase subunit alpha (ATP1A) Major histocompatibility complex class I-related gene protein (MR1) T-cell receptor alpha chain V region (TCRA) Probable G-protein coupled receptor 22 (GPR22) T-cell receptor beta chain (TRB) Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 (ASAP1) LanC-like protein 1 (LANCL1) Integral membrane protein GPR137C Coiled-coil domain-containing protein 47 (CCDC47) Cyclic nucleotide-gated olfactory channel (CNGA2) Solute carrier family 12 member 2 (SLC12A2) Integrin alpha-M (ITGAM)
Multi-pass Multi-pass Multi-pass Multi-pass
Hydroxycarboxylic acid receptor 1 (HCAR1) Interferon omega-1 (IFNW1)
membrane membrane membrane membrane
Secreted
a
MHC-I and MHC-II binding prediction results; bin the prediction method of the IEDB, percentile ranks go from 0 to 100. Since low percentile ranks are good binders, only those with a percentile rank < 5 were included in the BLAST analysis.
Table 3 Analysis of the possible homology of the predicted epitopes in Table 2 for HHV-6A-MCP with other human herpesviruses (only with E values < 0.1) Peptide
HHV-1
HHV-2
HHV-3
HHV-4
HHV-5
HHV-6B
HHV-7
HHV-8
FTQHLSFVDMGL LLSIMTLAAM NLFSAFPIHAFT IMIGNIPLPLAPV IVQRIDFADILPSV QLCGEPLV VISEYCMKSNSCL SLTSGSYPELAYV KLNDTMENNLPTSV GQQAICEVILTPV CLLYSSRAATSII DMMESV TESI RIDFADILPSV SLQLTFFFPLGIYIP TTLRRYYQPIPFHRF
– – – – – – – – – – – – – – –
– – – – – – – – – – – – – – –
– – – – – – – – – – – – – – –
– – – – – – – – – 0.021 – – – – –
– – – – – – – – – 0.040 – – – – –
0.0003 0.08 0.0006 0.0005 0.00005 – 0.00009 0.0003 0.000005 0.00009 0.0005 0.02 0.01 0.0000002 0.00000002
– – – 0.021 – – – – 0.00003 0.009 0.029 – – – 0.025
– – – – – – – – – – – – – – –
that they were present in three PPMS patients (37% of this group) but only in one (14%) with the RRMS form. Although these preliminary results should be confirmed in a larger series, they suggest a closer
association of HHV-6 and the PPMS form. In this line, a recent study reported the presence of DNA HHV-6 in the brain of MS patients, and particularly in progressive forms [26]. © 2014 The Author(s) European Journal of Neurology © 2014 EAN
Major HHV-6 antigen recognized by CSF IgG in MS
In summary, this work identifies MCP as the major antigen recognized specifically by IgG purified from the CSF of MS patients. Future studies will show if these antibodies play a role in the pathogenesis of the disease.
Acknowledgements This work was supported by grants PI12/02349 and PI12/00239 from the Instituto de Salud Carlos III (Spain) and grant SAF2012-34670 from the Ministerio de Economıa y Competitividad (Spain). Roberto Alvarez-Lafuente is the recipient of a research contract of the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (Feder) (CP07/00273). Raquel Alenda is the recipient of a research contract of the Red Espa~ nola de Esclerosis M ultiple (REEM) from the Instituto de Salud Carlos III (Spain).
Disclosure of conflicts of interest The authors declare no financial or other conflicts of interest.
References 1. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995; 92: 7440–7444. 2. Merelli E, Bedin R, Sola P, et al. Human herpes virus 6 and human herpes virus 8 DNA sequences in brains of multiple sclerosis patients, normal adults and children. J Neurol 1997; 244: 450–454. 3. Mayne M, Krishnan J, Metz L, et al. Infrequent detection of human herpesvirus 6 DNA in peripheral blood mononuclear cells from multiple sclerosis patients. Ann Neurol 1998; 44: 391–394. 4. Cuomo L, Trivedi P, Cardillo MR, et al. Human herpesvirus 6 infection in neoplastic and normal brain tissue. J Med Virol 2001; 63: 45–51. 5. Friedman JE, Lyons MJ, Cu G, et al. The association of the human herpesvirus-6 and MS. Mult Scler 1999; 5: 355–362. 6. Xu Y, Linde A, Fredrikson S, Dahl H, Winberg G. HHV-6 A- or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum. J Med Virol 2002; 66: 394–399. 7. Tejada-Simon MV, Zang YC, Hong J, Rivera VM, Killian JM, Zhang JZ. Detection of viral DNA and immune responses to the human herpesvirus 6 101kilodalton virion protein in patients with multiple sclerosis and in controls. J Virol 2002; 76: 6147–6154. 8. Al-Shammari S, Nelson RF, Voevodin A. HHV-6 DNAaemia in patients with multiple sclerosis in Kuwait. Acta Neurol Scand 2003; 107: 122–124. 9. Chapenko S, Millers A, Nora Z, Logina I, Kukaine R, Murovska M. Correlation between HHV-6 reactivation and multiple sclerosis disease activity. J Med Virol 2003; 69: 111–117.
© 2014 The Author(s) European Journal of Neurology © 2014 EAN
1101
10. Garcia-Montojo M, Martinez A, De Las Heras V, et al. Herpesvirus active replication in multiple sclerosis. A genetic control? J Neurol Sci 2011; 311: 98–102. 11. Garcia-Montojo M, De Las Heras V, Dominguez-Mozo M, et al. Human herpesvirus 6 and effectiveness of interferon beta 1b in multiple sclerosis patients. Eur J Neurol 2011; 18: 1027–1035. 12. Berti R, Brennan MB, Soldan SS, et al. Increased detection of serum HHV-6 DNA sequences during multiple sclerosis (MS) exacerbations and correlation with parameters of MS disease progression. J Neurovirol 2002; 8: 250–256. 13. Simpson S Jr, Taylor B, Dwyer DE. Anti-HHV-6 IgG titer significantly predicts subsequent relapse risk in multiple sclerosis. Mult Scler 2012; 18: 799–806. 14. Derfuss T, Hohlfeld R, Meinl E. 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. J Neurol 2005; 252: 968–971. 15. Virtanen JO, F€ arkkil€ a M, Multanen J, et al. Evidence for human herpesvirus 6 variant A antibodies in multiple sclerosis: diagnostic and therapeutic implications. J. Neurovirol 2007; 13: 347–352. 16. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis. Ann. Neurol 2001; 50: 121–127. 17. S adaba MC, Gonz alez Porque P, Masjuan J, AlvarezCerme~ no JC, Bootello A, Villar LM. An ultrasensitive method for the detection of oligoclonal IgG bands. J Immunol Methods 2004; 284: 141–145. 18. Peters B, Sidney J, Bourne P, et al. The design and implementation of the immune epitope database and analysis resource. Immunogenetics 2005; 57: 326–336. 19. Peters B, Sidney J, Bourne P, et al. The immune epitope database and analysis resource: from vision to blueprint. PLoS Biol 2005; 3: e91. 20. Lassmann H, Bruck W, Lucchinetti CF. The immunopathology of multiple sclerosis: an overview. Brain Pathol 2007; 17: 210–218. 21. Virtanen J, Wohler J, Fenton K, Reich DS, Jacobson S. Oligoclonal bands in multiple sclerosis reactive against two herpesviruses and association with magnetic resonance imaging findings. Mult Scler 2014; 20: 27–34. 22. Virtanen JO, Pietil€ ainen-Nicklen J, Uotila L, F€ arkkil€ a M, Vaheri A, Koskiniemi M. Intrathecal human herpesvirus 6 antibodies in multiple sclerosis and other demyelinating diseases presenting as oligoclonal bands in cerebrospinal fluid. J Neuroimmunol 2011; 237: 93–97. 23. Fotheringham J, Jacobson J. Human herpesvirus 6 and multiple sclerosis: potential mechanisms for virusinduced disease. Herpes 2005; 12: 4–9. 24. Littler E, Lawrence G, Liu MY, Barrell BG, Arrand JR. Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6. J Virol 1990; 64: 714–722. 25. Mitsukawa K, Lu X, Bartfai T. Galanin, galanin receptors and drug targets. Cell Mol Life Sci 2008; 5: 1796– 1805. 26. Mancuso R, Hernis A, Cavarretta R, et al. Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis. J Med Virol 2010; 82: 1051–1057.
