Brains and peripheral blood mononuclear cells of multiple sclerosis (MS) patients hyperexpress...

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Brains and peripheral blood mononuclear cells ofmultiple sclerosis (MS) patients hyperexpressMS-associated retrovirus/HERV-W endogenousretrovirus, but not Human herpesvirus 6

Giuseppe Mameli,1 Vito Astone,1 Giannina Arru,2 Silvia Marconi,3

Laura Lovato,3 Caterina Serra,1 Stefano Sotgiu,2 Bruno Bonetti3

and Antonina Dolei1

Correspondence

Antonina Dolei

[email protected]

1Section of Microbiology, Department of Biomedical Sciences, Center of Excellence forBiotechnology Development and Biodiversity Research, University of Sassari, Sassari, Italy

2Institute of Clinical Neurology, University of Sassari, Sassari, Italy

3Section of Neurology, Department of Neurological Sciences and Vision, University of Verona,Verona, Italy

Received 31 January 2006

Accepted 25 September 2006

Multiple sclerosis (MS)-associated retrovirus (MSRV)/HERV-W (human endogenous retrovirus W)

and Human herpesvirus 6 (HHV-6) are the two most studied (and discussed) viruses as

environmental co-factors that trigger MS immunopathological phenomena. Autopsied brain tissues

from MS patients and controls and peripheral blood mononuclear cells (PBMCs) were analysed.

Quantitative RT-PCR and PCR with primers specific for MSRV/HERV-W env and pol and HHV-6

U94/rep and DNA-pol were used to determine virus copy numbers. Brain sections were

immunostained with HERV-W env-specific monoclonal antibody to detect the viral protein. All

brains expressed MSRV/HERV-W env and pol genes. Phylogenetic analysis indicated that cerebral

MSRV/HERV-W-related env sequences, plasmatic MSRV, HERV-W and ERVWE1 (syncytin) are

related closely. Accumulation of MSRV/HERV-W-specific RNAs was significantly greater in MS

brains than in controls (P=0.014 vs healthy controls; P=0.006 vs pathological controls). By

immunohistochemistry, no HERV-W env protein was detected in control brains, whereas it was

upregulated within MS plaques and correlated with the extent of active demyelination and

inflammation. No HHV-6-specific RNAs were detected in brains of MS patients; one healthy control

had latent HHV-6 and one pathological control had replicating HHV-6. At the PBMC level, all MS

patients expressed MSRV/HERV-W env at higher copy numbers than did controls (P=0.00003).

Similar HHV-6 presence was found in MS patients and healthy individuals; only one MS patient

had replicating HHV-6. This report, the first to study both MSRV/HERV-W and HHV-6, indicates

that MSRV/HERV-W is expressed actively in human brain and activated strongly in MS patients,

whilst there are no significant differences between these MS patients and controls for HHV-6

presence/replication at the brain or PBMC level.

INTRODUCTION

The aetiopathogenesis of multiple sclerosis (MS) disease iscomplex and debated. Immunopathogenic phenomena arethought to be triggered by environmental (viral?) factorsoperating on a predisposing genetic background(Noseworthy et al., 2000). Among the viruses suggested asMS co-factors are ubiquitous members of the familyHerpesviridae, Human herpesvirus 6 (HHV-6) (Alvarez-Lafuente et al., 2004; Moore & Wolfson, 2002) and

Epstein–Barr virus (EBV) (Christensen, 2006), and ahuman endogenous retrovirus (HERV), the MS-associatedretrovirus (MSRV) (Dolei, 2005; Perron et al., 1989), amember of the HERV-W multicopy family; links betweenHERVs and some human diseases have been observedincreasingly (Dolei, 2006). HHV-6 can be neurotropic, canbecome latent and be reactivated, and has potentialimmunopathogenic properties. Meta-analyses indicatethat the available reports provide some support for a linkbetween HHV-6 and MS, but none shows causativerelationships (Clark, 2004; Moore & Wolfson, 2002). Theassociation of MS with EBV is based on the increasedfrequency of late infectious mononucleosis, higher titres of

A supplementary table showing primers used in this study is available inJGV Online.

264 0008-1890 G 2007 SGM Printed in Great Britain

Journal of General Virology (2007), 88, 264–274 DOI 10.1099/vir.0.81890-0


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anti-EBV EBNA IgG and the almost absolute prevalence oflatent EBV infection in MS patients (Delorenze et al., 2006;Hollsberg et al., 2005). MSRV/HERV-W produces extra-cellular virus particles with gliotoxic (apoptotic) (Menardet al., 1997), fusogenic (Perron et al., 1997a) and super-antigenic (Perron et al., 2001) properties; the virus causes aT cell-mediated neuropathology in vivo (Firouzi et al.,2003). Activities strikingly concordant with findings onMSRV and MS (Dolei et al., 2002; Firouzi et al., 2003; Lafonet al., 2002; Perron et al., 2005; Sotgiu et al., 2002b) werereported for syncytin (Antony et al., 2004), an env proteinencoded by the ERVWE1 locus (Blond et al., 1999) andinvolved in embryo implantation during pregnancy (Miet al., 2000; Muir et al., 2004); this element is a replication-incompetent endogenous retrovirus belonging to theHERV-W family, located on chromosome 7q21–22, in aregion of candidate genetic susceptibility for MS (Blaiseet al., 2003). Moreover, a complex interplay might occurbetween HHV-6 and MSRV/HERV-W, as MSRV expressionis transactivated in vitro by herpesviruses (Lafon et al., 2002;Perron et al., 1993) and the simultaneous presence of HERVand herpesvirus antigens has synergistic effects on cell-mediated immune responses (Brudek et al., 2004).

To date, MSRV virions have been detected in the blood andcerebrospinal fluid (CSF) of MS patients by several groups,including ourselves (Dolei et al., 2002; Komurian-Pradelet al., 1999; Nowak et al., 2003; Perron et al., 1997b; Serraet al., 2001; Sotgiu et al., 2002b). The virus is also detectablein other neurological patients (Dolei et al., 2002; Karlssonet al., 2004; Nowak et al., 2003), at significantly lowerfrequencies, and in about 10 % of healthy individuals(Dolei et al., 2002; Garson et al., 1998). In a populationat high risk of MS, we detected MSRV particles in theplasma of 100 % of patients with active MS, and the presenceof MSRV particles in CSF was found to parallel theprogression of the disease (Dolei et al., 2002). Notably, blindserial examinations revealed that patients with MSRV-freeCSF had stable MS, whereas those with MSRV-positive CSFdisclosed a more severe, treatment-requiring disease,suggesting that the presence of MSRV in CSF could beconsidered as a negative prognostic marker (Sotgiu et al.,2002b, 2007). Accordingly, MSRV presence in CSF ofmonosymptomatic optic neuritis patients is associated withincreased conversion to definite MS (Sotgiu et al., 2006).

To provide some insight into the role of these two viruses inMS, we evaluated their expression in brain and blood cells ofMS patients and controls, as the mere detection of theirgenomes per se would not imply viral activity. This isparticularly important for HHV-6 studies, as the vastmajority of reports have detected virus DNA, and the needfor studies to examine antigen and virus mRNA expressionin MS and control brains to delineate the relationshipbetween latent and active virus and MS has recently beenunderlined (Fotheringham & Jacobson, 2005). We evaluatedtwo different transcripts for each virus [MSRV/HERV-Wenv and pol, encoding envelope and reverse transcriptase

(RT), respectively; HHV-6 U94/rep, which maintains thelatent state, and DNA-pol, indicative of actively replicatingvirus]. To our knowledge, this is the first study to evaluateboth MSRV/HERV-W and HHV-6 and the first to use a fullyquantitative approach to determine MSRV/HERV-W load.

METHODS

Human samples. Early post-mortem (mean interval, 8.5 h; range,4–12 h) brain tissue from five subjects (mean age, 48 years; range,37–65 years) with a clinical diagnosis of chronic progressive MS wasstudied. In total, 14 blocks containing lesions and normal-appearingwhite matter were examined. Histopathologically, cases MS1–MS3

had predominantly chronic-active lesions with hypercellular mar-gins, ongoing demyelination with macrophage and lymphocyte infil-tration and a hypocellular demyelinated centre. In cases MS4 andMS5, the majority of lesions were chronic-silent (absence of inflam-mation and presence of demyelinated centres). Controls (Table 1)consisted of four normal brain controls (NBCs), autopsied tissuefrom subjects with non-neurological diseases (mean age, 45 years;range, 37–60 years; post-mortem mean interval, 12.2 h; range,8–20 h), six patients with other neurological diseases (OND), autop-sied tissue from cases OND7 and OND8 (Alzheimer’s disease, age66 years; cerebral infarct, age 65 years) and bioptic samples fromfour patients with grade IV astrocytoma (mean age, 45.5 years;range, 42–52 years), including neoplastic and normal peritumoraltissue. In all autopsied cases, sampling provided cortical structuresand white matter from frontal and parietal lobes. All samples weresnap-frozen and stored at 280 uC, and 15 mm thick cryostat sectionsunderwent immunohistochemistry. Samples for RNA extractionwere from chronic-active MS lesions and/or periplaque tissue: greyand white matter from NBCs, astrocytomas (OND1–OND4), peritu-moral (OND5) and normal tissue (OND6) surrounding sampleOND1 and frontal grey matter from OND7 and OND8. Integrity ofbrain samples was controlled by immunohistochemistry (see below).We could analyse only a small number of brain samples, as it is noteasy to obtain freshly frozen brain samples. Brain samples employedin this study were already available [obtained by one of us (B. B.) fordiagnostic purposes over the past 5 years]; therefore, ethical approvalwas not necessary.

Blood samples from 35 patients with active MS and 14 healthy donors(HDs; blood donors from a transfusion centre) were derived from acohort described previously (Table 2) (Dolei et al., 2002).

RNA extraction. Peripheral blood mononuclear cell (PBMC)separation and RNA extraction were described previously (Doleiet al., 2002; Serra et al., 2003). Thawed brain fragments were washedwith PBS to remove blood trails and then RNA extraction was per-formed as described above.

RT-PCR for MSRV/HERV-W pol RNA. RNA-derived cDNA sam-ples were exposed to nested qualitative and non-nested semi-quanti-tative RT-PCR using MSRV/HERV-W pol-specific primers, asdescribed previously (Dolei et al., 2002; Perron et al., 1997b; Serraet al., 2003). Briefly, 100 ng aliquots of undiluted RNA sample oreach of a series of serial dilutions underwent reverse transcriptioninto DNA by using oligo-dT as primer and M-MLV (Moloneymurine leukemia virus) RT (Gibco-BRL Life Technologies) asdescribed previously (Dolei et al., 2002), followed by PCR amplifica-tion of DNA products in a Hybaid thermal cycler (Omnigene) utiliz-ing primers specific for the MSRV/HERV-W pol gene (Dolei et al.,2002; Garson et al., 1998). Controls included PCR of RNAs notexposed to RT with primers specific for the b-globin gene (primerpair PC04/GH20; Synthetic Genetics) or with MSRV/HERV-W-spe-cific primers (to ensure the absence of contaminating cellular DNA

http://vir.sgmjournals.org 265

MSRV/HERV-W and HHV-6 in multiple sclerosis


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sequences and of endogenous retroviral DNA sequences, respec-tively), PCR of cDNA samples without template (negative control)and samples of human cellular DNA (positive control). CellularRNA from PBMCs of individuals shown previously to be negativefor circulating MSRV (and whose PBMCs did not release or tran-scribe MSRV/HERV-W in culture; Serra et al., 2003) was alsoincluded. Presence/absence of MSRV/HERV-W was confirmed inrepeated assays of the same sample. The specificity of the amplifiedproducts was confirmed by dideoxy sequencing (see below). Semi-quantitative data were expressed as the reciprocal of each end-pointMSRV/HERV-W-positive dilution in semi-quantitative RT-PCR.

Quantitative real-time RT-PCR for MSRV/HERV-W env,HHV-6 DNA-pol and HHV-6 U94/rep RNAs and generation ofrecombinant DNA external calibration curves. Primers andTaqMan probes were designed by using Beacon Designer software(PREMIER Biosoft International) (see Supplementary Table S1,available in JGV Online). HHV-6 primers and probes recognizeboth HHV-6A and HHV-6B variants [GenBank accession numbersX83413 (HHV-6A) and AF157706 (HHV-6B)]. Samples were heatedto 95 uC for 3 min and then subjected to 50 cycles of 94 uC for 15 s,53 uC for 30 s and 60 uC for 30 s in an iCycler iQ PCR detectionsystem (Bio-Rad). Fluorescent data were collected during the 60 uCstep. Each sample was analysed at least twice and the specific contentwas obtained through external calibration curves; gene fragments ofinterest, generated by conventional PCR, were inserted into plasmids(pCR2.1-TOPO; Invitrogen) as standards for the production ofrecombinant DNA (rDNA) (Pfaffl & Hageleit, 2001). We used

rDNA standards instead of RNAs for RNA quantification as theygive better results in terms of sensitivity, quantification range, repro-ducibility and stability (Pfaffl & Hageleit, 2001) and DNA standardshave also been used in a recent paper on the development of broadlytargeted real-time PCRs for semi-quantification of HERV pol RNAexpression (Forsman et al., 2005). To control for correct amplifica-tion and reverse transcription, a positive sample was also submittedto each RNA-extraction procedure and the resulting extract wasamplified in triplicate. Parallel RNA samples were also exposed toPCR amplification without the RT step to detect contaminatingDNA. Prevention measures against cross-contamination wereemployed (Kwok & Higuchi, 1989); in particular, sample processingand PCR amplification were carried out in separate laboratories,with different equipment.

Phylogenetic analysis of MSRV/HERV-W env sequences.Amplified products were obtained by RT-PCR amplification utiliz-ing primers specific for MSRV/HERV-W env (see SupplementaryTable S1, available in JGV Online). The size of amplified productscovered almost one-third of the env RNA sequence, including theintracellular region. Amplified products were then exposed to auto-mated dideoxy sequencing in both directions, with the fluorescentBigDye system (Perkin Elmer ABI PRISM 310 Genetic Analyzer).Sequence analysis was carried out by using CLUSTAL_X (Thompsonet al., 1997) for multiple sequence alignment and UPGMA(unweighted pair-group method using averages) for phylogeneticanalysis. Sequences from patients and controls were compared withthose of other HERV env sequences [GenBank accession numbers

Table 1. MSRV/HERV-W and HHV-6 expression in human brains from MS patients and controls

MS, Multiple sclerosis patients; OND, patients with other neurological diseases; NBC, normal brain controls.

Group Brain sample MSRV/HERV-W HHV-6

pol* envD U94/repD DNA-polD

MSd MS1 Periphery + 81 920 2 2

MS2 Periphery + 71 570 2 2

MS3 Plaque + 105 550 2 2

Mean±SD, 89 555±15 601 2 2

NBC§ NBC1 White matter + 3 824 23 2

NBC1 Grey matter + 6 144 22 2

NBC2 Grey matter + 926 2 2

NBC3 Grey matter + 8 016 2 2

NBC4 White matter + 1 986 2 2

Mean±SD, 4 179±291 9.0±12.3 2

OND|| OND1 Astrocytoma + 4 787 2 2

OND2 Astrocytoma + 3 856 2 2

OND3 Astrocytoma + 413 2 2

OND4 Astrocytoma + 3 440 2 2

OND5 Peritumoral + 3 454 2 2

OND6 Normal + 460 2 10

OND7 Stroke + 141 2 2

OND8 Alzheimer’s + 22 2 2

Mean±SD, 2 072±1 986 2 1.2±3.5

*Positivity in nested RT-PCR.

DCopies per 100 ng RNA.

dMS vs NBCs, Kruskal–Wallis H P=0.014.

§NBCs vs OND, not significant.

||MS vs OND, P=0.006.

266 Journal of General Virology 88

G. Mameli and others


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AF331500 (MSRV virionic genome), NM_014590 (ERVWE1-syncytin mRNA), AJ289710 (HERV-H), Y18890 (HERV-K) andNM_207582 (HERV-FRD/syncytin2)].

Immunohistochemistry. The anti-HERV-W env monoclonal anti-body (mAb) 6A2B2 (Blond et al., 2000) was employed in bothimmunoperoxidase and double-immunofluorescence procedures toassess HERV-W reactivity in brain tissues on each MS block, on 12blocks from NBCs (three blocks for each patient) and on each ONDsample. Procedures were described in detail elsewhere (Bonetti et al.,2003; Lolli et al., 2005). Immunoperoxidase staining was performedin duplicate and reactivity was graded as follows: 2, no staining; +,staining present on <20 % of cells (four representative fields at406 magnification for each case); 2+, staining on 20–40 % of cells;3+, staining on 40–60 % of cells; 4+, staining present on >60 %of cells. Slides were viewed under a Zeiss MC80 microscope. For

double fluorescence, anti-HERV-W reactivity was detected withstreptavidin–Texas red (Amersham Biosciences); sections were thenincubated with the glial fibrillary acidic protein (GFAP) phenotypicmarker (1 : 500; Dako) followed by fluorescein-conjugated anti-rabbit Ig or anti-HLA-DR (1 : 10; Dako). To assess the cellular distri-bution in brain sections, double immunofluorescence with theneural phenotypic markers myelin basic protein (MBP; 1 : 200) formature oligodendrocytes, GFAP (1 : 500) for astrocytes, CD68for microglia (1 : 100) (all from Dako) and NG2 (1 : 100; Chemicon)for oligodendrocyte precursors was performed as described pre-viously (Lolli et al., 2005). The reaction was visualized with appro-priate fluorescein-conjugated antibodies (Vector Laboratories).

Statistical analysis. Significance of the results was evaluated bymeans of the Epi Info database and statistics software program, ver-sion 6 (CDC/WHO, Atlanta, GA, USA).

Table 2. MSRV/HERV-W and HHV-6 presence/expression in PBMCs from MS patients and healthy blood donors

HD, Healthy blood donors; MS, patients with active MS; NS, not significant; NA, not applicable.

HD MS

All HHV-6+ high* HHV-6+ lowD All HHV-6+ high HHV-6+ low

No. 14 0 4 35 1 11

Age (years):

Mean±SD 32.3±9.2 40.1±8.1 37.1±9.9 28 36.1±10.4

Range 19–52 33–52 19–52 27.4–50.2

Median 32 37.5 36 36

P valued vs HD NS NA NS

MSRV/HERV-W env RNA

No. positive/total 4/14 – 2/3 35/35 1/1 11/11

P value§ vs HD <0.000001 NA NS

No. copies per 100 ng cell RNA:

Mean 890.5±2.791 – 352.5±523.1 4580.4±7158.8 24400 3418.4±3254.1

Range 0–10 520 0–953.5 169–26 450 436–9890

Median 0|| 1540 1877

P valued vs HD 0.00003 NA 0.02

HHV-6 DNA-pol DNA

No. positive/total 3/14 0 3/3 12/35 1/1 11/11

P value§ vs HD NS NA NS


Mean 8.6±17.4 0 40.4±6.6 982.0±5539.8 32811 141.6±204.6

Range 0–47.4 34.2–47.4 0–32 811 0–689

Median 0 104.0 0 51.4


HHV-6 DNA-pol RNA

No. positive/total 0/14 – 0 1/35 1/1 0/11


Mean 0 0 19.5±113.9 664 0

Range 0–0 0–664 0–0

Median 0 0 0


*High copy numbers of HHV-6 DNA-pol DNA, i.e. actively replicating HHV-6.

DFewer than 400 HHV-6 DNA copies.

dKruskal–Wallis H P value.

§Two-tailed Fisher’s exact test.

||Fewer than 10 copies.




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RESULTS

Detection of HERV-W RNA transcripts and inter-relationships between the known HERV-W envsequences

All brain samples tested contained HERV-W env and polRNA transcripts, irrespective of the health/disease status(Table 1); MSRV/HERV-W env was detected in PBMCsfrom 35 of 35 MS patients and four of 14 HDs (Table 2), inkeeping with data of extracellular MSRV (Dolei et al., 2002).Samples of MSRV/HERV-W env products, obtained bynested RT-PCR amplification of brain RNAs from MSpatients, were sequenced in comparison with env sequencesof extracellular virus from plasma of MS patients and HDindividuals from a previously described cohort (Dolei et al.,2002) of the Italian island Sardinia (which has the second-highest MS prevalence worldwide) (Sotgiu et al., 2002a), aswell as to other HERV-W sequences present in GenBank,such as that of extracellular genomic MSRV (Perron et al.,2001) and of ERVWE1 (syncytin) (Mi et al., 2000) mRNA,and to env sequences from other endogenous and exogenoushuman retroviruses. For the sequence under study, i.e. theenv intracellular domain, MSRV and ERVWE1 env RNAshave >89 % identity, and the env consensus sequenceof extracellular genomic MSRV/HERV-W detected inSardinian bloods shares >95 and >93 % identity withMSRV and ERVWE1, respectively (if the whole env gene iscompared, MSRV and ERVWE1 env RNAs have >93 %identity and the Sardinian env consensus sequence shares>95 % identity with both MSRV and ERVWE1; data notshown), thus indicating that all of these genes arehomologous to each other and that, until now, no specificprimers have been identified to discriminate env sequencesof extracellular MSRV from those possibly transcribed fromother HERV-W endogenous DNAs, such as the ERVWE1locus. A computer-derived phylogenetic tree of the above

sequences is shown in Fig. 1. As shown, all sequences arerelated closely and belong to the same branch. As for theextent of identity in the gene fragment of Fig. 1, the envsequences detected in brain tissues from the MS patientsshare up to 96 % identity with the MSRV, ERVWE1 andSardinian MSRV/HERV-W consensus sequence. All of theenv sequences detected in our human samples belongunequivocally to the HERV-W family and are distant fromother HERV families (38–47 % identity with HERV-FRD/syncytin 2, HERV-H and HERV-K) and from exogenoushuman retroviruses, such as human T-lymphotropic virus 1and human immunodeficiency virus (20–25 % identity; datanot shown).

MSRV/HERV-W env and pol genes areupregulated in MS patients

Quantification of MSRV/HERV-W env and pol RNAtranscripts in brain and PBMC samples was carried outby real-time RT-PCR and semi-quantitative RT-PCR,respectively. Data are reported in Tables 1 and 2 andFig. 2. As shown, MSRV/HERV-W env transcripts accu-mulated similarly in brain samples from NBCs and ONDcontrols, without significant differences between white andgrey matter. In brain samples from MS patients, however,the accumulation of MSRV/HERV-W transcripts wasincreased by 20- to 25-fold. Notably, very similar increaseswere obtained in MS patients by testing two differentMSRV/HERV-W genes and by using two different techni-ques (env, 21.4-fold by quantitative real-time RT-PCR; pol,23.3-fold by semi-quantitative RT-PCR) and the differencebetween MS and controls was highly significant(Table 1; Fig. 2). In PBMCs, MSRV/HERV-W env RNAcopy numbers were higher in MS patients than incontrols (P=0.00003; Table 2), due to the increasedfrequency of MSRV/HERV-W positivity, as the differencein MSRV/HERV-W env RNA copy numbers between

HERV-K

HERV-HHERV-FRD

HD1

HD2MSRVSYNHD4

MS1

MS4

0.1

HD

3

ONDMS

3

SARD-CONSMS2

Fig. 1. Computer-derived phylogenetic tree ofMSRV/HERV-W env sequences. Thesequences of MSRV/HERV-W env amplifiedproducts from brain tissues were analysed asdescribed in Methods and compared phylo-genetically with known env sequences presentin GenBank from other human endogenousretroviruses [accession nos: MSRV,AF331500; ERVWE1-syncytin (SYN),NM_014590; HERV-H env, AJ289710;HERV-K env, Y18890; HERV-FRD/syncytin2,NM_207582], extracellular MSRV from plasmaof MS patients and healthy donors (HDs) froman already described cohort (Dolei et al.,2002) and the Sardinian consensus sequence(SARD-CONS). MS, MS patients (MS1–2,blood; MS3–4, brain); OND, patients with otherneurological diseases; SARD-CONS, consen-sus sequence from plasmas of the Sardiniancohort.




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MSRV/HERV-W-positive MS and healthy individuals wasnot significant (data not shown).

MSRV/HERV-W env protein is detectable in thebrain of MS patients, but not in normal brain

The cellular distribution of brain sections and the integrityof autopsied samples, in comparison with biopsied ones,were assessed through immunofluorescence detection ofneural phenotypic markers, such as MBP for matureoligodendrocytes, GFAP for astrocytes, CD68 for microgliaand NG2 for oligodendrocyte precursors (Lolli et al., 2005).These markers did not show quantitative differences in thenormal-appearing white matter of MS (autopsied) brainswith respect to either autopsied or biopsied normal-appearing or OND brains (data not shown).

No staining by the HERV-W env 6A2B2 mAb (Blond et al.,2000) was detected in either grey or white matter in brainfrom NBCs (Fig. 3a) or from Alzheimer’s disease patients,or in brain tissue surrounding malignant astrocytoma(Fig. 3b), whilst scattered glial cells within neoplastic lesionsshowed MSRV/HERV-W immunoreactivity (Fig. 3c). InMS lesions, upregulation of MSRV/HERV-W immunor-eactivity was observed within plaques, correlated with theextent of active demyelination and inflammation. In fact,MSRV/HERV-W immunoreactivity was absent in normal-appearing white matter and in perilesional areas (notshown). In chronic-silent MS lesions, very faint MSRV/HERV-W staining, located at the lesion edge on a limitedproportion of glial cells (Fig. 3e), was observed in three ofseven plaques (1+); in the remaining silent lesions, nostaining was detected throughout. In chronic-active MSlesions, MSRV/HERV-W immunoreactive cells were abun-dant (40–60 % of total glial cells, 3+) and presentthroughout the entire lesion. In terms of localization onglial-cell subpopulations, MSRV/HERV-W immunostain-ing was observed on cells resembling both astrocytes andmicroglia at the lesion edge (Fig. 3f), as described recently(Antony et al., 2004; Perron et al., 2005); this finding wasconfirmed by double immunofluorescence, where theMSRV/HERV-W signal co-localized either with GFAP- or

HLA-DR-positive cells (data not shown). At variance withactive lesion edges, in plaque centres, the MSRV/HERV-Wsignal was mostly localized on hypertrophic astrocytes(Fig. 3g and insert).

Lack of expression of HHV-6 sequences inbrain and PBMCs of MS patients

Given the ubiquitous distribution of HHV-6 infection inhumans, two different real-time RT-PCRs were performedto discriminate between actively replicating virus (expres-sing DNA-pol RNA transcripts) and latent virus, which it isknown to express only the U94/rep gene to maintain latency(Yoshikawa et al., 2002). As reported in Table 1, brainsamples from MS patients had levels of both HHV-6transcripts that were below detection limits (similar to thoseof other reports: Griscelli et al., 2001; Pfaffl & Hageleit,2001). One NBC had latent HHV-6 and one OND hadreplicating HHV-6; however, copy numbers were close todetection limits. This indicates that, at variance with studiesfrom other populations, HHV-6 is expressed only by a smallminority (if any) of brain samples from our country, none ofthem with MS.

As for PBMCs (Table 2), 21.4 % of HDs and 34.3 % of MSpatients had HHV-6 DNA. Virus DNA copies were slightlymore abundant in MS patients than in HDs, and one MSindividual had replicating HHV-6, as judged by detection ofDNA-pol transcripts. However, differences of neitherpercentage HHV-6 positivity nor DNA copy numbersreached statistical significance. In the MS cohort, meanMSRV/HERV-W copy numbers did not differ significantlyin HHV-6-positive and HHV-6-negative patients (P=0.13,Kruskal–Wallis test).

DISCUSSION

Virus involvement in MS pathogenesis is a highly debatedissue. According to the literature, the most likely candidateviruses as MS co-factors are herpesviruses, such as HHV-6(Alvarez-Lafuente et al., 2004; Moore & Wolfson, 2002) andEBV (Christensen, 2006; Delorenze et al., 2006; Hollsberg

MS

RV

/HE

RV

-W e

nv

MS

RV

/HE

RV

-W p

ol

100 000

80 000

60 000

40 000

20 000

(a) (b)250

200

150

50

100

* * * * * *

NBC OND MS NBC MS

Fig. 2. Upregulation of MSRV/HERV-W env

and pol gene expression in the brain of MSpatients. (a) MSRV/HERV-W env-specifictranscripts accumulated in brain samplesfrom patients and controls. Data areexpressed as mean env copies per 100 ngRNA, evaluated by real-time RT-PCR. *MSvs NBCs, Kruskal–Wallis H P=0.014; **MSvs ONDs, P=0.006. (b) MSRV/HERV-Wpol-specific transcripts; data are expressedas reciprocals of the end point of MSRV/HERV-W pol-positive dilutions in semi-quan-titative RT-PCR. ***P=0.017.




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et al., 2005), and endogenous retroviruses, such as MSRV/HERV-W (Dolei, 2005; Perron et al., 1989). Qualitative/semi-quantitative differences between patients and controlsfor presence/expression of either MSRV/HERV-W or HHV-6 have been reported (Christensen, 2005; Clark, 2004; Doleiet al., 2002; Garson et al., 1998; Komurian-Pradel et al.,1999; Moore & Wolfson, 2002; Nowak et al., 2003; Opsahl &Kennedy, 2005; Perron et al., 1997b; Yi et al., 2004). Thepresent study shows, for the first time, data on thesimultaneous expression of both viruses in PBMCs andbrain from MS patients and controls; they are strengthenedby quantification of the expression of two genes for eachvirus, because, given their ubiquity, genome detection per sewould not necessarily imply viral activity.

The origin of MS-associated HERV-W-related transcripts isdebated (Dolei, 2005; Garson et al., 2005): it could beexpression of isolated genes, such as syncytin (Mi et al.,2000), extracellular MSRV endogenous retrovirus particles

(Firouzi et al., 2003) or a new HERV-W exogenous memberof the HERV-W family (Dolei, 2005; Serra et al., 2003).However, no primers or antibodies are available fordiscriminating virion-producing/pathogenic MSRV/HERV-W from RNA or proteins normally expressed byendogenous HERV-W proviruses (Perron et al., 2005), andall known HERV-W env sequences are homologous (Fig. 1),including sequences present in GenBank or detectedexperimentally by us in brain tissues (that could be eitherintracellular RNAs or extracellular genomic RNAs), as wellas in plasma samples of patients and controls (extracellular,virionic genomes).

In all brain samples tested, we detected the presence of bothMSRV/HERV-W env and pol RNAs, regardless of the health/disease status of the individual. However, a statisticallysignificant increase in expression was observed in MSpatients with respect to those with normal-appearing brainsor with other neurological diseases (whose reciprocal

(a)

(e) (f) (g)

(b) (c) (d)

Fig. 3. Immunoreactivity for MSRV/HERV-W in control brains and MS lesions. Brain sections were processed as describedpreviously (Bonetti et al., 2003) and stained with the anti-HERV-W env 6A2B2 mAb (Blond et al., 2000). No immunostainingwas detected in normal white matter from normal brain controls (a) or in brain tissue surrounding malignant astrocytoma (b),whereas scattered glial cells within neoplastic lesions showed MSRV/HERV-W immunoreactivity (c). Immunostaining wasabsent in a serial section of the same neoplastic tissue as in (b), but stained with an isotype-matched unrelated antibody (d).In chronic-silent MS lesions, a very faint signal was present at the lesion edge (e). Intense signal was observed in chronic-active MS lesions (MS2), being localized on cells morphologically resembling microglia and astrocytes at the lesion edge (f),whereas in the plaque core, MSRV/HERV-W staining mainly decorated astrocytic profiles (g and insert). Magnification, 6240.




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differences were minor and not significant); the increase wassimilar for both genes, despite their evaluation with differentassays (env, 21.4-fold by quantitative real-time RT-PCR; pol,23.3-fold by semi-quantitative RT-PCR). This suggests a co-ordinated accumulation of the two transcripts, as occurs forgenes located close together. Quantitative real-time datasuggest that brains from MS patients and controls have aviral load of approximately 105 and 103.5 MSRV/HERV-Wenv copies per 100 ng RNA, respectively.

When serial sections of the same brain sample were analysedat the protein level (Fig. 3), the MSRV/HERV-W envprotein was clearly detected only in samples from MSpatients, perhaps due to lower sensitivity of this assaycompared with PCR methodology. Alternatively, one mightassume that MSRV/HERV-W RNA expression in thehealthy brain is not followed by protein synthesis (mostHERVs are defective and/or lacking 39 untranslated-regionsequences that are necessary to stabilize RNA translation ineukaryotic cells), whilst in MS lesions (and in scattered glialcells within the tumour), MSRV/HERV-W env has acquiredfeatures that allow the process of translation.Immunostaining was found only within MS lesions andits intensity correlated with active demyelination andinflammation, whereas normal-appearing white matterfrom the same patients was found to be negative. Inchronic-silent lesions, very faint staining for MSRV/HERVenv was present on a minority of glial cells, whilst in chronic-active MS plaques, immunoreactive cells were abundantthroughout the entire lesion. Around 50 % of total glial cellscontained the env protein. As for glial-cell subpopulations,at the active lesion edge, env immunoreactivity was observedon cells morphologically resembling both astrocytes andmicroglia; in plaque centres, instead, the MSRV/HERV-Wsignal was mostly localized on hypertrophic astrocytes.These findings are in keeping with studies of MS brains(Antony et al., 2004; Perron et al., 2005) showing relativeaccumulation of HERV-W env RNA and protein in brainfrom MS patients.

We also detected MSRV/HERV-W env and pol RNAs innormal brains, whilst MSRV/HERV-W env protein wasabsent in samples from normal brain controls and fromAlzheimer’s disease patients, as well as normal peritumoraltissues, but present in scattered glial cells within the tumour,thus confirming in vivo HERV-W expression found incancer cell lines (Yi et al., 2004). In the interpretation of datafrom various diseases, one should remember that weevidenced a positive-feedback loop on MSRV expressionin blood cells from MSRV-positive individuals (Serra et al.,2003); these cells release MSRV spontaneously in culture,which can be upregulated by exposure to the MS detrimentalcytokines gamma interferon (IFN-c) and tumour necrosisfactor alpha (TNF-a), whilst IFN-b, a therapeutic cytokinefor MS, is a powerful inhibitor of MSRV release. In keepingwith this, HERV-W RNA was detected in patients withAlzheimer’s disease only in the presence of TNF-a (Johnstonet al., 2001).

With respect to HHV-6, the other MS co-factor candidate,we could not detect HHV-6 expression in brain tissues ofMS patients (Table 1), despite the use of fully quantitativereal-time RT-PCRs that allow detection of both HHV-6 Aand B variants and discriminate between actively replicatingand latent virus. One normal NBC had latent HHV-6 andone pathological brain control had replicating HHV-6, butin copy numbers close to detection limits (which arecomparable to those of other real-time PCR studies)(Donati et al., 2003); similar viral loads were found in astudy of various brain regions from English MS cases andcontrols, without differences in the distribution, varianttype or quantity of HHV-6 in brains from patients with MScompared with controls (Tuke et al., 2004). In contrast, ahigher prevalence in the MS brain was found for HHV-6DNA in an American cohort (Cermelli et al., 2003) and forHHV-6 antigen in a Finnish study (Virtanen et al., 2005).Similarly, HHV-6 DNA was found in all early MS lesionsfrom five patients, albeit with scarce production of virusantigens, if any (Goodman et al., 2003). It was also suggestedthat HHV-6 might be more relevant in early than inestablished MS disease (Rotola et al., 2004). A recent papershowed HHV-6 expression in all brain samples of seven MSpatients and three controls of an English cohort, restricted tooligodendrocytes, whose percentage positivity was signifi-cantly higher in MS patients than in controls (Opsahl &Kennedy, 2005). Two meta-analyses have been performed onthe association of HHV-6 and MS (Clark, 2004; Moore &Wolfson, 2002), including studies on brain, CSF, blood etc.Among their conclusions is that, as HHV-6 is detected in ahigh proportion of individuals without MS, HHV-6 PCRpositivity in itself is not sufficient for its causality in thedevelopment of MS; the available reports provide somesupport for a relationship between HHV-6 and MS, but noneare able to show a causative relationship, and studies ofprevalence of HHV-6 infection do not provide conclusiveevidence for HHV-6–MS association. A recent review ofpotential HHV-6-induced disease mechanisms in MS under-lined the need for studies of antigen and virus mRNAexpression in the brain, to delineate the relationship betweenlatent and active virus and MS (Fotheringham & Jacobson,2005). It has also been hypothesized that the dysregulatedimmune system of MS patients is unable to control periodicHHV-6 flare-ups, which possibly contribute to MS pathol-ogy, where HHV-6 infection might affect neural-cell function(Alvarez-Lafuente et al., 2004; Fotheringham & Jacobson,2005; Opsahl & Kennedy, 2005). To our knowledge, only tworeports have been published so far on HHV-6 RNAexpression in MS, in the brain (Opsahl & Kennedy, 2005)and in the blood (Alvarez-Lafuente et al., 2004).

In our opinion, both HERV-W and HHV-6 have thepotential for a role in MS. However, MSRV/HERV-W is/arethought to be present in all human cells, whereas HHV-6,although specific seroreactivity is acquired very early byhumans, can be variably present in the nervous system,according to its circulation/reactivation in different popula-tions and cohorts. This could explain the wide differences




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observed in the various studies (Bonetti et al., 2003; Clark,2004; Fotheringham & Jacobson, 2005; Moore & Wolfson,2002). In addition, herpesviruses might activate HERVexpression in patients, as MSRV expression can betransactivated in vitro by herpesviruses (Lafon et al., 2002;Perron et al., 1993) and the simultaneous presence of HERVand herpesvirus antigens has pronounced synergistic effectson cell-mediated immune responses (Brudek et al., 2004).Christensen (2005) recently reviewed the possible interac-tions between HERV and herpesvirus, proposing tosynergize the herpesvirus and HERV findings, and presentedseveral possible pathogenic mechanisms.

In conclusion, our study shows the co-ordinated expressionof MSRV/HERV-W pol and env in all brain samples, which isincreased by 1.4 logs in MS tissues, where the env protein isalso detectable. The data exclude actual HHV-6 involvementin the MS lesions under study and provide very limitedevidence of brain infection by HHV-6 in our controls. Thissuggests either that HHV-6 acts very early during MS or thatits reportedly increased presence is an epiphenomenon,deriving from the activation of a pre-existing latent virus inthe brain. From the bulk of published reports, it appears thatHHV-6, as with other herpesviruses, has the potential totransactivate MSRV/HERV-W (Brudek et al., 2004; Lafonet al., 2002) and to exert pathogenic phenomena on braintissues (Christensen, 2005; Fotheringham & Jacobson, 2005).Nonetheless, in the MS lesions studied, we found MSRV/HERV-W activation in the absence of latent or replicatingHHV-6. Our data from blood cells from a wider cohortreinforce the data from brains. In fact, even though thepresence of HHV-6 DNA occurs in a minority of individuals,with a slight, but not significant, increase in MS PBMCs, wedetected HHV-6 RNA expression only in one MS sample andin no controls (2.8 and 0 %, respectively; P>0.05, notsignificant). It must be pointed out that the presence ofHHV-6 DNA in the healthy has been reported to range from0 to 60 % in different populations (Fotheringham &Jacobson, 2005), therefore explaining discordant reports(Alvarez-Lafuente et al., 2004; Cermelli et al., 2003; Clark,2004; Goodman et al., 2003; Moore & Wolfson, 2002; Opsahl& Kennedy, 2005; Rotola et al., 2004; Tuke et al., 2004).

Our conclusion is that the correlation between MS andincreased HHV-6 presence/expression in brain and PBMCsis not a general finding (Clark, 2004; Fotheringham &Jacobson, 2005; Moore & Wolfson, 2002); on the contrary,MSRV/HERV-W was found to be activated by us and in allstudies of MS brain and blood tissues (Antony et al., 2004;Dolei et al., 2002; Garson et al., 1998; Johnston et al., 2001;Nowak et al., 2003; Perron et al., 1997a, 2005; Sotgiu et al.,2002b), and could be a new target of therapy (Antony et al.,2004; Dolei, 2005; Serra et al., 2003).

ACKNOWLEDGEMENTS

Work was supported partly by grants from Fondazione Italiana SclerosiMultipla Onlus (grant no. 2005/R/11), Ministero Universita e Ricerca

and Regione Autonoma Sardegna. We thank L. Poddighe for PBMCRNA extraction. G. M. is a PhD fellow of the European Community.The anti- HERV-W env 6A2B2 mAb was provided by bioMerieux.

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