Journal of General Virology (2007), 88, 264–274
DOI 10.1099/vir.0.81890-0
Brains and peripheral blood mononuclear cells of
multiple sclerosis (MS) patients hyperexpress
MS-associated retrovirus/HERV-W endogenous
retrovirus, 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]
1
Section of Microbiology, Department of Biomedical Sciences, Center of Excellence for
Biotechnology Development and Biodiversity Research, University of Sassari, Sassari, Italy
2
Institute of Clinical Neurology, University of Sassari, Sassari, Italy
3
Section 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 is
complex and debated. Immunopathogenic phenomena are
thought to be triggered by environmental (viral?) factors
operating on a predisposing genetic background
(Noseworthy et al., 2000). Among the viruses suggested as
MS co-factors are ubiquitous members of the family
Herpesviridae, Human herpesvirus 6 (HHV-6) (AlvarezLafuente et al., 2004; Moore & Wolfson, 2002) and
A supplementary table showing primers used in this study is available in
JGV Online.
264
Epstein–Barr virus (EBV) (Christensen, 2006), and a
human endogenous retrovirus (HERV), the MS-associated
retrovirus (MSRV) (Dolei, 2005; Perron et al., 1989), a
member of the HERV-W multicopy family; links between
HERVs and some human diseases have been observed
increasingly (Dolei, 2006). HHV-6 can be neurotropic, can
become latent and be reactivated, and has potential
immunopathogenic properties. Meta-analyses indicate
that the available reports provide some support for a link
between HHV-6 and MS, but none shows causative
relationships (Clark, 2004; Moore & Wolfson, 2002). The
association of MS with EBV is based on the increased
frequency of late infectious mononucleosis, higher titres of
Downloaded from www.microbiologyresearch.org by
0008-1890 G 2007 SGM
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Printed in Great Britain
MSRV/HERV-W and HHV-6 in multiple sclerosis
anti-EBV EBNA IgG and the almost absolute prevalence of
latent EBV infection in MS patients (Delorenze et al., 2006;
Hollsberg et al., 2005). MSRV/HERV-W produces extracellular virus particles with gliotoxic (apoptotic) (Menard
et al., 1997), fusogenic (Perron et al., 1997a) and superantigenic (Perron et al., 2001) properties; the virus causes a
T cell-mediated neuropathology in vivo (Firouzi et al.,
2003). Activities strikingly concordant with findings on
MSRV and MS (Dolei et al., 2002; Firouzi et al., 2003; Lafon
et al., 2002; Perron et al., 2005; Sotgiu et al., 2002b) were
reported for syncytin (Antony et al., 2004), an env protein
encoded by the ERVWE1 locus (Blond et al., 1999) and
involved in embryo implantation during pregnancy (Mi
et al., 2000; Muir et al., 2004); this element is a replicationincompetent endogenous retrovirus belonging to the
HERV-W family, located on chromosome 7q21–22, in a
region of candidate genetic susceptibility for MS (Blaise
et al., 2003). Moreover, a complex interplay might occur
between HHV-6 and MSRV/HERV-W, as MSRV expression
is transactivated in vitro by herpesviruses (Lafon et al., 2002;
Perron et al., 1993) and the simultaneous presence of HERV
and herpesvirus antigens has synergistic effects on cellmediated immune responses (Brudek et al., 2004).
To date, MSRV virions have been detected in the blood and
cerebrospinal fluid (CSF) of MS patients by several groups,
including ourselves (Dolei et al., 2002; Komurian-Pradel
et al., 1999; Nowak et al., 2003; Perron et al., 1997b; Serra
et al., 2001; Sotgiu et al., 2002b). The virus is also detectable
in other neurological patients (Dolei et al., 2002; Karlsson
et al., 2004; Nowak et al., 2003), at significantly lower
frequencies, and in about 10 % of healthy individuals
(Dolei et al., 2002; Garson et al., 1998). In a population
at high risk of MS, we detected MSRV particles in the
plasma of 100 % of patients with active MS, and the presence
of MSRV particles in CSF was found to parallel the
progression of the disease (Dolei et al., 2002). Notably, blind
serial examinations revealed that patients with MSRV-free
CSF had stable MS, whereas those with MSRV-positive CSF
disclosed a more severe, treatment-requiring disease,
suggesting that the presence of MSRV in CSF could be
considered as a negative prognostic marker (Sotgiu et al.,
2002b, 2007). Accordingly, MSRV presence in CSF of
monosymptomatic optic neuritis patients is associated with
increased conversion to definite MS (Sotgiu et al., 2006).
(RT), respectively; HHV-6 U94/rep, which maintains the
latent state, and DNA-pol, indicative of actively replicating
virus]. To our knowledge, this is the first study to evaluate
both MSRV/HERV-W and HHV-6 and the first to use a fully
quantitative 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 was
studied. In total, 14 blocks containing lesions and normal-appearing
white matter were examined. Histopathologically, cases MS1–MS3
had predominantly chronic-active lesions with hypercellular margins, ongoing demyelination with macrophage and lymphocyte infiltration and a hypocellular demyelinated centre. In cases MS4 and
MS5, the majority of lesions were chronic-silent (absence of inflammation and presence of demyelinated centres). Controls (Table 1)
consisted of four normal brain controls (NBCs), autopsied tissue
from 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), autopsied tissue from cases OND7 and OND8 (Alzheimer’s disease, age
66 years; cerebral infarct, age 65 years) and bioptic samples from
four patients with grade IV astrocytoma (mean age, 45.5 years;
range, 42–52 years), including neoplastic and normal peritumoral
tissue. In all autopsied cases, sampling provided cortical structures
and white matter from frontal and parietal lobes. All samples were
snap-frozen and stored at 280 uC, and 15 mm thick cryostat sections
underwent immunohistochemistry. Samples for RNA extraction
were from chronic-active MS lesions and/or periplaque tissue: grey
and white matter from NBCs, astrocytomas (OND1–OND4), peritumoral (OND5) and normal tissue (OND6) surrounding sample
OND1 and frontal grey matter from OND7 and OND8. Integrity of
brain samples was controlled by immunohistochemistry (see below).
We could analyse only a small number of brain samples, as it is not
easy to obtain freshly frozen brain samples. Brain samples employed
in this study were already available [obtained by one of us (B. B.) for
diagnostic purposes over the past 5 years]; therefore, ethical approval
was not necessary.
Blood samples from 35 patients with active MS and 14 healthy donors
(HDs; blood donors from a transfusion centre) were derived from a
cohort described previously (Table 2) (Dolei et al., 2002).
RNA extraction. Peripheral blood mononuclear cell (PBMC)
separation and RNA extraction were described previously (Dolei
et al., 2002; Serra et al., 2003). Thawed brain fragments were washed
with PBS to remove blood trails and then RNA extraction was performed as described above.
RT-PCR for MSRV/HERV-W pol RNA. RNA-derived cDNA sam-
To provide some insight into the role of these two viruses in
MS, we evaluated their expression in brain and blood cells of
MS patients and controls, as the mere detection of their
genomes per se would not imply viral activity. This is
particularly important for HHV-6 studies, as the vast
majority of reports have detected virus DNA, and the need
for studies to examine antigen and virus mRNA expression
in MS and control brains to delineate the relationship
between latent and active virus and MS has recently been
underlined (Fotheringham & Jacobson, 2005). We evaluated
two different transcripts for each virus [MSRV/HERV-W
env and pol, encoding envelope and reverse transcriptase
http://vir.sgmjournals.org
ples were exposed to nested qualitative and non-nested semi-quantitative RT-PCR using MSRV/HERV-W pol-specific primers, as
described previously (Dolei et al., 2002; Perron et al., 1997b; Serra
et al., 2003). Briefly, 100 ng aliquots of undiluted RNA sample or
each of a series of serial dilutions underwent reverse transcription
into DNA by using oligo-dT as primer and M-MLV (Moloney
murine leukemia virus) RT (Gibco-BRL Life Technologies) as
described previously (Dolei et al., 2002), followed by PCR amplification of DNA products in a Hybaid thermal cycler (Omnigene) utilizing primers specific for the MSRV/HERV-W pol gene (Dolei et al.,
2002; Garson et al., 1998). Controls included PCR of RNAs not
exposed to RT with primers specific for the b-globin gene (primer
pair PC04/GH20; Synthetic Genetics) or with MSRV/HERV-W-specific primers (to ensure the absence of contaminating cellular DNA
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
265
G. Mameli and others
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
81 920
71 570
105 550
Mean±SD, 89 555±15 601
3 824
6 144
926
8 016
1 986
Mean±SD, 4 179±291
4 787
3 856
413
3 440
3 454
460
141
22
Mean±SD, 2 072±1 986
2
2
2
2
23
22
2
2
2
9.0±12.3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10
2
2
1.2±3.5
MSd
MS1
MS2
MS3
Periphery
Periphery
Plaque
+
+
+
NBC§
NBC1
NBC1
NBC2
NBC3
NBC4
White matter
Grey matter
Grey matter
Grey matter
White matter
+
+
+
+
+
OND||
OND1
OND2
OND3
OND4
OND5
OND6
OND7
OND8
Astrocytoma
Astrocytoma
Astrocytoma
Astrocytoma
Peritumoral
Normal
Stroke
Alzheimer’s
+
+
+
+
+
+
+
+
*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.
sequences and of endogenous retroviral DNA sequences, respectively), PCR of cDNA samples without template (negative control)
and samples of human cellular DNA (positive control). Cellular
RNA from PBMCs of individuals shown previously to be negative
for circulating MSRV (and whose PBMCs did not release or transcribe MSRV/HERV-W in culture; Serra et al., 2003) was also
included. Presence/absence of MSRV/HERV-W was confirmed in
repeated assays of the same sample. The specificity of the amplified
products was confirmed by dideoxy sequencing (see below). Semiquantitative data were expressed as the reciprocal of each end-point
MSRV/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 of
recombinant DNA external calibration curves. Primers and
TaqMan probes were designed by using Beacon Designer software
(PREMIER Biosoft International) (see Supplementary Table S1,
available in JGV Online). HHV-6 primers and probes recognize
both HHV-6A and HHV-6B variants [GenBank accession numbers
X83413 (HHV-6A) and AF157706 (HHV-6B)]. Samples were heated
to 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 detection
system (Bio-Rad). Fluorescent data were collected during the 60 uC
step. Each sample was analysed at least twice and the specific content
was obtained through external calibration curves; gene fragments of
interest, generated by conventional PCR, were inserted into plasmids
(pCR2.1-TOPO; Invitrogen) as standards for the production of
recombinant DNA (rDNA) (Pfaffl & Hageleit, 2001). We used
266
rDNA standards instead of RNAs for RNA quantification as they
give better results in terms of sensitivity, quantification range, reproducibility and stability (Pfaffl & Hageleit, 2001) and DNA standards
have also been used in a recent paper on the development of broadly
targeted real-time PCRs for semi-quantification of HERV pol RNA
expression (Forsman et al., 2005). To control for correct amplification and reverse transcription, a positive sample was also submitted
to each RNA-extraction procedure and the resulting extract was
amplified in triplicate. Parallel RNA samples were also exposed to
PCR amplification without the RT step to detect contaminating
DNA. Prevention measures against cross-contamination were
employed (Kwok & Higuchi, 1989); in particular, sample processing
and 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 utilizing primers specific for MSRV/HERV-W env (see Supplementary
Table S1, available in JGV Online). The size of amplified products
covered almost one-third of the env RNA sequence, including the
intracellular region. Amplified products were then exposed to automated dideoxy sequencing in both directions, with the fluorescent
BigDye system (Perkin Elmer ABI PRISM 310 Genetic Analyzer).
Sequence analysis was carried out by using CLUSTAL_X (Thompson
et al., 1997) for multiple sequence alignment and UPGMA
(unweighted pair-group method using averages) for phylogenetic
analysis. Sequences from patients and controls were compared with
those of other HERV env sequences [GenBank accession numbers
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Journal of General Virology 88
MSRV/HERV-W and HHV-6 in multiple sclerosis
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
No.
Age (years):
Mean±SD
Range
Median
P valued vs HD
MSRV/HERV-W env RNA
No. positive/total
P value§ vs HD
No. copies per 100 ng cell RNA:
Mean
Range
Median
P valued vs HD
HHV-6 DNA-pol DNA
No. positive/total
P value§ vs HD
No. copies per 100 ng cell RNA:
Mean
Range
Median
P valued vs HD
HHV-6 DNA-pol RNA
No. positive/total
No. copies per 100 ng cell RNA:
Mean
Range
Median
P valued vs HD
MS
All
HHV-6+ high*
HHV-6+ lowD
All
HHV-6+ high
HHV-6+ low
14
0
4
35
1
11
40.1±8.1
33–52
37.5
37.1±9.9
19–52
36
28
36.1±10.4
27.4–50.2
36
NS
NA
NS
35/35
<0.000001
1/1
11/11
NA
NS
4580.4±7158.8
169–26 450
1540
0.00003
24400
NA
3418.4±3254.1
436–9890
1877
0.02
12/35
1/1
11/11
NS
NA
NS
982.0±5539.8
0–32 811
0
32811
141.6±204.6
0–689
51.4
NS
NA
NS
0
1/35
1/1
0/11
0
19.5±113.9
0–664
0
664
0
0–0
0
NS
NA
NS
32.3±9.2
19–52
32
4/14
890.5±2.791
0–10 520
0||
3/14
8.6±17.4
0–47.4
0
0/14
–
–
0
0
–
0
0–0
0
2/3
352.5±523.1
0–953.5
3/3
40.4±6.6
34.2–47.4
104.0
*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.
AF331500 (MSRV virionic genome), NM_014590 (ERVWE1syncytin mRNA), AJ289710 (HERV-H), Y18890 (HERV-K) and
NM_207582 (HERV-FRD/syncytin2)].
Immunohistochemistry. The anti-HERV-W env monoclonal anti-
body (mAb) 6A2B2 (Blond et al., 2000) was employed in both
immunoperoxidase and double-immunofluorescence procedures to
assess HERV-W reactivity in brain tissues on each MS block, on 12
blocks from NBCs (three blocks for each patient) and on each OND
sample. Procedures were described in detail elsewhere (Bonetti et al.,
2003; Lolli et al., 2005). Immunoperoxidase staining was performed
in duplicate and reactivity was graded as follows: 2, no staining; +,
staining present on <20 % of cells (four representative fields at
406 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
http://vir.sgmjournals.org
double fluorescence, anti-HERV-W reactivity was detected with
streptavidin–Texas red (Amersham Biosciences); sections were then
incubated with the glial fibrillary acidic protein (GFAP) phenotypic
marker (1 : 500; Dako) followed by fluorescein-conjugated antirabbit Ig or anti-HLA-DR (1 : 10; Dako). To assess the cellular distribution in brain sections, double immunofluorescence with the
neural phenotypic markers myelin basic protein (MBP; 1 : 200) for
mature oligodendrocytes, GFAP (1 : 500) for astrocytes, CD68
for microglia (1 : 100) (all from Dako) and NG2 (1 : 100; Chemicon)
for oligodendrocyte precursors was performed as described previously (Lolli et al., 2005). The reaction was visualized with appropriate fluorescein-conjugated antibodies (Vector Laboratories).
Statistical analysis. Significance of the results was evaluated by
means of the Epi Info database and statistics software program, version 6 (CDC/WHO, Atlanta, GA, USA).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
267
G. Mameli and others
RESULTS
Detection of HERV-W RNA transcripts and interrelationships between the known HERV-W env
sequences
RD
-C
ON
S
MS2
Quantification of MSRV/HERV-W env and pol RNA
transcripts in brain and PBMC samples was carried out
by real-time RT-PCR and semi-quantitative RT-PCR,
respectively. Data are reported in Tables 1 and 2 and
Fig. 2. As shown, MSRV/HERV-W env transcripts accumulated similarly in brain samples from NBCs and OND
controls, without significant differences between white and
grey matter. In brain samples from MS patients, however,
the accumulation of MSRV/HERV-W transcripts was
increased by 20- to 25-fold. Notably, very similar increases
were obtained in MS patients by testing two different
MSRV/HERV-W genes and by using two different techniques (env, 21.4-fold by quantitative real-time RT-PCR; pol,
23.3-fold by semi-quantitative RT-PCR) and the difference
between MS and controls was highly significant
(Table 1; Fig. 2). In PBMCs, MSRV/HERV-W env RNA
copy numbers were higher in MS patients than in
controls (P=0.00003; Table 2), due to the increased
frequency of MSRV/HERV-W positivity, as the difference
in MSRV/HERV-W env RNA copy numbers between
HD
HERV-K
HD 3D
ON S 3
M
SA
MSRV/HERV-W env and pol genes are
upregulated in MS patients
4
All brain samples tested contained HERV-W env and pol
RNA transcripts, irrespective of the health/disease status
(Table 1); MSRV/HERV-W env was detected in PBMCs
from 35 of 35 MS patients and four of 14 HDs (Table 2), in
keeping with data of extracellular MSRV (Dolei et al., 2002).
Samples of MSRV/HERV-W env products, obtained by
nested RT-PCR amplification of brain RNAs from MS
patients, were sequenced in comparison with env sequences
of extracellular virus from plasma of MS patients and HD
individuals from a previously described cohort (Dolei et al.,
2002) of the Italian island Sardinia (which has the secondhighest MS prevalence worldwide) (Sotgiu et al., 2002a), as
well 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 exogenous
human retroviruses. For the sequence under study, i.e. the
env intracellular domain, MSRV and ERVWE1 env RNAs
have >89 % identity, and the env consensus sequence
of extracellular genomic MSRV/HERV-W detected in
Sardinian bloods shares >95 and >93 % identity with
MSRV and ERVWE1, respectively (if the whole env gene is
compared, MSRV and ERVWE1 env RNAs have >93 %
identity and the Sardinian env consensus sequence shares
>95 % identity with both MSRV and ERVWE1; data not
shown), thus indicating that all of these genes are
homologous to each other and that, until now, no specific
primers have been identified to discriminate env sequences
of extracellular MSRV from those possibly transcribed from
other HERV-W endogenous DNAs, such as the ERVWE1
locus. A computer-derived phylogenetic tree of the above
sequences is shown in Fig. 1. As shown, all sequences are
related closely and belong to the same branch. As for the
extent of identity in the gene fragment of Fig. 1, the env
sequences detected in brain tissues from the MS patients
share up to 96 % identity with the MSRV, ERVWE1 and
Sardinian MSRV/HERV-W consensus sequence. All of the
env sequences detected in our human samples belong
unequivocally to the HERV-W family and are distant from
other HERV families (38–47 % identity with HERV-FRD/
syncytin 2, HERV-H and HERV-K) and from exogenous
human retroviruses, such as human T-lymphotropic virus 1
and human immunodeficiency virus (20–25 % identity; data
not shown).
N
SY SRV
M 2
HD
MS4
MS1
HD1
0.1
268
HERV-H
HERV-FRD
Fig. 1. Computer-derived phylogenetic tree of
MSRV/HERV-W
env
sequences.
The
sequences of MSRV/HERV-W env amplified
products from brain tissues were analysed as
described in Methods and compared phylogenetically with known env sequences present
in GenBank from other human endogenous
retroviruses
[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 plasma
of MS patients and healthy donors (HDs) from
an 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 other
neurological diseases; SARD-CONS, consensus sequence from plasmas of the Sardinian
cohort.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Journal of General Virology 88
MSRV/HERV-W and HHV-6 in multiple sclerosis
250
(a)
(b)
200
80 000
MSRV/HERV-W pol
MSRV/HERV-W env
100 000
60 000
40 000
150
100
50
20 000
*
* *
NBC
OND
* * *
MS
NBC
MSRV/HERV-W-positive MS and healthy individuals was
not significant (data not shown).
MSRV/HERV-W env protein is detectable in the
brain of MS patients, but not in normal brain
The cellular distribution of brain sections and the integrity
of autopsied samples, in comparison with biopsied ones,
were assessed through immunofluorescence detection of
neural phenotypic markers, such as MBP for mature
oligodendrocytes, GFAP for astrocytes, CD68 for microglia
and NG2 for oligodendrocyte precursors (Lolli et al., 2005).
These markers did not show quantitative differences in the
normal-appearing white matter of MS (autopsied) brains
with respect to either autopsied or biopsied normalappearing 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 brain
from NBCs (Fig. 3a) or from Alzheimer’s disease patients,
or in brain tissue surrounding malignant astrocytoma
(Fig. 3b), whilst scattered glial cells within neoplastic lesions
showed MSRV/HERV-W immunoreactivity (Fig. 3c). In
MS lesions, upregulation of MSRV/HERV-W immunoreactivity was observed within plaques, correlated with the
extent of active demyelination and inflammation. In fact,
MSRV/HERV-W immunoreactivity was absent in normalappearing white matter and in perilesional areas (not
shown). In chronic-silent MS lesions, very faint MSRV/
HERV-W staining, located at the lesion edge on a limited
proportion of glial cells (Fig. 3e), was observed in three of
seven plaques (1+); in the remaining silent lesions, no
staining was detected throughout. In chronic-active MS
lesions, MSRV/HERV-W immunoreactive cells were abundant (40–60 % of total glial cells, 3+) and present
throughout the entire lesion. In terms of localization on
glial-cell subpopulations, MSRV/HERV-W immunostaining was observed on cells resembling both astrocytes and
microglia at the lesion edge (Fig. 3f), as described recently
(Antony et al., 2004; Perron et al., 2005); this finding was
confirmed by double immunofluorescence, where the
MSRV/HERV-W signal co-localized either with GFAP- or
http://vir.sgmjournals.org
MS
Fig. 2. Upregulation of MSRV/HERV-W env
and pol gene expression in the brain of MS
patients. (a) MSRV/HERV-W env-specific
transcripts accumulated in brain samples
from patients and controls. Data are
expressed as mean env copies per 100 ng
RNA, evaluated by real-time RT-PCR. *MS
vs NBCs, Kruskal–Wallis H P=0.014; **MS
vs ONDs, P=0.006. (b) MSRV/HERV-W
pol-specific transcripts; data are expressed
as reciprocals of the end point of MSRV/
HERV-W pol-positive dilutions in semi-quantitative RT-PCR. ***P=0.017.
HLA-DR-positive cells (data not shown). At variance with
active lesion edges, in plaque centres, the MSRV/HERV-W
signal was mostly localized on hypertrophic astrocytes
(Fig. 3g and insert).
Lack of expression of HHV-6 sequences in
brain and PBMCs of MS patients
Given the ubiquitous distribution of HHV-6 infection in
humans, two different real-time RT-PCRs were performed
to discriminate between actively replicating virus (expressing DNA-pol RNA transcripts) and latent virus, which it is
known to express only the U94/rep gene to maintain latency
(Yoshikawa et al., 2002). As reported in Table 1, brain
samples from MS patients had levels of both HHV-6
transcripts that were below detection limits (similar to those
of other reports: Griscelli et al., 2001; Pfaffl & Hageleit,
2001). One NBC had latent HHV-6 and one OND had
replicating HHV-6; however, copy numbers were close to
detection limits. This indicates that, at variance with studies
from other populations, HHV-6 is expressed only by a small
minority (if any) of brain samples from our country, none of
them with MS.
As for PBMCs (Table 2), 21.4 % of HDs and 34.3 % of MS
patients had HHV-6 DNA. Virus DNA copies were slightly
more abundant in MS patients than in HDs, and one MS
individual had replicating HHV-6, as judged by detection of
DNA-pol transcripts. However, differences of neither
percentage HHV-6 positivity nor DNA copy numbers
reached statistical significance. In the MS cohort, mean
MSRV/HERV-W copy numbers did not differ significantly
in HHV-6-positive and HHV-6-negative patients (P=0.13,
Kruskal–Wallis test).
DISCUSSION
Virus involvement in MS pathogenesis is a highly debated
issue. According to the literature, the most likely candidate
viruses as MS co-factors are herpesviruses, such as HHV-6
(Alvarez-Lafuente et al., 2004; Moore & Wolfson, 2002) and
EBV (Christensen, 2006; Delorenze et al., 2006; Hollsberg
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
269
G. Mameli and others
(a)
(e)
(b)
(c)
(f)
(d)
(g)
Fig. 3. Immunoreactivity for MSRV/HERV-W in control brains and MS lesions. Brain sections were processed as described
previously (Bonetti et al., 2003) and stained with the anti-HERV-W env 6A2B2 mAb (Blond et al., 2000). No immunostaining
was 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 was
absent 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 chronicactive 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.
et al., 2005), and endogenous retroviruses, such as MSRV/
HERV-W (Dolei, 2005; Perron et al., 1989). Qualitative/
semi-quantitative differences between patients and controls
for presence/expression of either MSRV/HERV-W or HHV6 have been reported (Christensen, 2005; Clark, 2004; Dolei
et 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). The
present study shows, for the first time, data on the
simultaneous expression of both viruses in PBMCs and
brain from MS patients and controls; they are strengthened
by quantification of the expression of two genes for each
virus, because, given their ubiquity, genome detection per se
would not necessarily imply viral activity.
The origin of MS-associated HERV-W-related transcripts is
debated (Dolei, 2005; Garson et al., 2005): it could be
expression of isolated genes, such as syncytin (Mi et al.,
2000), extracellular MSRV endogenous retrovirus particles
270
(Firouzi et al., 2003) or a new HERV-W exogenous member
of the HERV-W family (Dolei, 2005; Serra et al., 2003).
However, no primers or antibodies are available for
discriminating
virion-producing/pathogenic
MSRV/
HERV-W from RNA or proteins normally expressed by
endogenous HERV-W proviruses (Perron et al., 2005), and
all known HERV-W env sequences are homologous (Fig. 1),
including sequences present in GenBank or detected
experimentally by us in brain tissues (that could be either
intracellular RNAs or extracellular genomic RNAs), as well
as in plasma samples of patients and controls (extracellular,
virionic genomes).
In all brain samples tested, we detected the presence of both
MSRV/HERV-W env and pol RNAs, regardless of the health/
disease status of the individual. However, a statistically
significant increase in expression was observed in MS
patients with respect to those with normal-appearing brains
or with other neurological diseases (whose reciprocal
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Journal of General Virology 88
MSRV/HERV-W and HHV-6 in multiple sclerosis
differences were minor and not significant); the increase was
similar for both genes, despite their evaluation with different
assays (env, 21.4-fold by quantitative real-time RT-PCR; pol,
23.3-fold by semi-quantitative RT-PCR). This suggests a coordinated accumulation of the two transcripts, as occurs for
genes located close together. Quantitative real-time data
suggest that brains from MS patients and controls have a
viral load of approximately 105 and 103.5 MSRV/HERV-W
env copies per 100 ng RNA, respectively.
When serial sections of the same brain sample were analysed
at the protein level (Fig. 3), the MSRV/HERV-W env
protein was clearly detected only in samples from MS
patients, perhaps due to lower sensitivity of this assay
compared with PCR methodology. Alternatively, one might
assume that MSRV/HERV-W RNA expression in the
healthy brain is not followed by protein synthesis (most
HERVs are defective and/or lacking 39 untranslated-region
sequences that are necessary to stabilize RNA translation in
eukaryotic cells), whilst in MS lesions (and in scattered glial
cells within the tumour), MSRV/HERV-W env has acquired
features that allow the process of translation.
Immunostaining was found only within MS lesions and
its intensity correlated with active demyelination and
inflammation, whereas normal-appearing white matter
from the same patients was found to be negative. In
chronic-silent lesions, very faint staining for MSRV/HERV
env was present on a minority of glial cells, whilst in chronicactive MS plaques, immunoreactive cells were abundant
throughout the entire lesion. Around 50 % of total glial cells
contained the env protein. As for glial-cell subpopulations,
at the active lesion edge, env immunoreactivity was observed
on cells morphologically resembling both astrocytes and
microglia; in plaque centres, instead, the MSRV/HERV-W
signal 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 relative
accumulation of HERV-W env RNA and protein in brain
from MS patients.
We also detected MSRV/HERV-W env and pol RNAs in
normal brains, whilst MSRV/HERV-W env protein was
absent in samples from normal brain controls and from
Alzheimer’s disease patients, as well as normal peritumoral
tissues, but present in scattered glial cells within the tumour,
thus confirming in vivo HERV-W expression found in
cancer cell lines (Yi et al., 2004). In the interpretation of data
from various diseases, one should remember that we
evidenced a positive-feedback loop on MSRV expression
in 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 detrimental
cytokines gamma interferon (IFN-c) and tumour necrosis
factor alpha (TNF-a), whilst IFN-b, a therapeutic cytokine
for MS, is a powerful inhibitor of MSRV release. In keeping
with this, HERV-W RNA was detected in patients with
Alzheimer’s disease only in the presence of TNF-a (Johnston
et al., 2001).
http://vir.sgmjournals.org
With respect to HHV-6, the other MS co-factor candidate,
we could not detect HHV-6 expression in brain tissues of
MS patients (Table 1), despite the use of fully quantitative
real-time RT-PCRs that allow detection of both HHV-6 A
and B variants and discriminate between actively replicating
and latent virus. One normal NBC had latent HHV-6 and
one pathological brain control had replicating HHV-6, but
in copy numbers close to detection limits (which are
comparable to those of other real-time PCR studies)
(Donati et al., 2003); similar viral loads were found in a
study of various brain regions from English MS cases and
controls, without differences in the distribution, variant
type or quantity of HHV-6 in brains from patients with MS
compared with controls (Tuke et al., 2004). In contrast, a
higher prevalence in the MS brain was found for HHV-6
DNA in an American cohort (Cermelli et al., 2003) and for
HHV-6 antigen in a Finnish study (Virtanen et al., 2005).
Similarly, HHV-6 DNA was found in all early MS lesions
from five patients, albeit with scarce production of virus
antigens, if any (Goodman et al., 2003). It was also suggested
that HHV-6 might be more relevant in early than in
established MS disease (Rotola et al., 2004). A recent paper
showed HHV-6 expression in all brain samples of seven MS
patients and three controls of an English cohort, restricted to
oligodendrocytes, whose percentage positivity was significantly higher in MS patients than in controls (Opsahl &
Kennedy, 2005). Two meta-analyses have been performed on
the 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 a
high proportion of individuals without MS, HHV-6 PCR
positivity in itself is not sufficient for its causality in the
development of MS; the available reports provide some
support for a relationship between HHV-6 and MS, but none
are able to show a causative relationship, and studies of
prevalence of HHV-6 infection do not provide conclusive
evidence for HHV-6–MS association. A recent review of
potential HHV-6-induced disease mechanisms in MS underlined the need for studies of antigen and virus mRNA
expression in the brain, to delineate the relationship between
latent and active virus and MS (Fotheringham & Jacobson,
2005). It has also been hypothesized that the dysregulated
immune system of MS patients is unable to control periodic
HHV-6 flare-ups, which possibly contribute to MS pathology, where HHV-6 infection might affect neural-cell function
(Alvarez-Lafuente et al., 2004; Fotheringham & Jacobson,
2005; Opsahl & Kennedy, 2005). To our knowledge, only two
reports have been published so far on HHV-6 RNA
expression 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 the
potential for a role in MS. However, MSRV/HERV-W is/are
thought to be present in all human cells, whereas HHV-6,
although specific seroreactivity is acquired very early by
humans, can be variably present in the nervous system,
according to its circulation/reactivation in different populations and cohorts. This could explain the wide differences
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
271
G. Mameli and others
observed in the various studies (Bonetti et al., 2003; Clark,
2004; Fotheringham & Jacobson, 2005; Moore & Wolfson,
2002). In addition, herpesviruses might activate HERV
expression in patients, as MSRV expression can be
transactivated in vitro by herpesviruses (Lafon et al., 2002;
Perron et al., 1993) and the simultaneous presence of HERV
and herpesvirus antigens has pronounced synergistic effects
on cell-mediated immune responses (Brudek et al., 2004).
Christensen (2005) recently reviewed the possible interactions between HERV and herpesvirus, proposing to
synergize the herpesvirus and HERV findings, and presented
several possible pathogenic mechanisms.
In conclusion, our study shows the co-ordinated expression
of MSRV/HERV-W pol and env in all brain samples, which is
increased by 1.4 logs in MS tissues, where the env protein is
also detectable. The data exclude actual HHV-6 involvement
in the MS lesions under study and provide very limited
evidence of brain infection by HHV-6 in our controls. This
suggests either that HHV-6 acts very early during MS or that
its reportedly increased presence is an epiphenomenon,
deriving from the activation of a pre-existing latent virus in
the brain. From the bulk of published reports, it appears that
HHV-6, as with other herpesviruses, has the potential to
transactivate MSRV/HERV-W (Brudek et al., 2004; Lafon
et al., 2002) and to exert pathogenic phenomena on brain
tissues (Christensen, 2005; Fotheringham & Jacobson, 2005).
Nonetheless, in the MS lesions studied, we found MSRV/
HERV-W activation in the absence of latent or replicating
HHV-6. Our data from blood cells from a wider cohort
reinforce the data from brains. In fact, even though the
presence of HHV-6 DNA occurs in a minority of individuals,
with a slight, but not significant, increase in MS PBMCs, we
detected HHV-6 RNA expression only in one MS sample and
in no controls (2.8 and 0 %, respectively; P>0.05, not
significant). It must be pointed out that the presence of
HHV-6 DNA in the healthy has been reported to range from
0 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 and
increased HHV-6 presence/expression in brain and PBMCs
is 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 all
studies 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).
REFERENCES
Alvarez-Lafuente, R., De las Heras, V., Bartolome, M., Picazo, J. J. &
Arroyo, R. (2004). Relapsing-remitting multiple sclerosis and human
herpesvirus 6 active infection. Arch Neurol 61, 1523–1527.
Antony, J. M., Van Marle, G., Opii, W., Butterfield, D. A., Mallet, F.,
Yong, V. W., Wallace, J. L., Deacon, R. M., Warren, K. & Power, C.
(2004). Human endogenous retrovirus glycoprotein-mediated induc-
tion of redox reactants causes oligodendrocyte death and demyelination. Nat Neurosci 7, 1088–1095.
Blaise, S., de Parseval, N., Benit, L. & Heidmann, T. (2003).
Genomewide screening for fusogenic human endogenous retrovirus
envelopes identifies syncytin 2, a gene conserved on primate
evolution. Proc Natl Acad Sci U S A 100, 13013–13018.
Blond, J. L., Beseme, F., Duret, L., Bouton, O., Bedin, F., Perron, H.,
Mandrand, B. & Mallet, F. (1999). Molecular characterisation and
placental expression of HERV-W, a new human endogenous
retrovirus family. J Virol 73, 1175–1185.
Blond, J. L., Lavillette, D., Cheynet, V., Bouton, O., Oriol, G.,
Chapel-Fernandes, S., Mandrand, B., Mallet, F. & Cosset, F. L.
(2000). An envelope glycoprotein of the human endogenous
retrovirus HERV-W is expressed in the human placenta and fuses
cells expressing the type D mammalian retrovirus receptor. J Virol
74, 3321–3329.
Bonetti, B., Valdo, P., Ossi, G., De Toni, L., Masotto, B., Marconi, S.,
Rizzuto, N., Nardelli, E. & Moretto, G. (2003). T-cell cytotoxicity of
human Schwann cells: TNFalpha promotes fasL-mediated apoptosis
and IFN gamma perforin-mediated lysis. Glia 43, 141–148.
Brudek, T., Christensen, T., Hansen, H. J., Bobecka, J. &
Moller-Larsen, A. (2004). Simultaneous presence of endogenous
retrovirus and herpes virus antigens has profound effect on cellmediated immune responses: implications for multiple sclerosis.
AIDS Res Hum Retroviruses 20, 415–423.
Cermelli, C., Berti, R., Soldan, S. S., Mayne, M., D’ambrosia, J. M.,
Ludwin, S. K. & Jacobson, S. (2003). High frequency of human
herpesvirus 6 DNA in multiple sclerosis plaques isolated by laser
microdissection. J Infect Dis 187, 1377–1387.
Christensen, T. (2005). Association of human endogenous retro-
viruses with multiple sclerosis and possible interactions with herpes
viruses. Rev Med Virol 15, 179–211.
Christensen, T. (2006). The role of EBV in MS pathogenesis. Int MS
J 13, 52–57.
Clark, D. (2004). Human herpesvirus type 6 and multiple sclerosis.
Herpes 11 (Suppl. 2), 112A–119A.
Delorenze, G. N., Munger, K. L., Lennette, E. T., Orentreich, N.,
Vogelman, J. H. & Ascherio, A. (2006). Epstein-Barr virus and
multiple sclerosis: evidence of association from a prospective study
with long-term follow-up. Arch Neurol 63, 839–844.
Dolei, A. (2005). MSRV/HERV-W/syncytin and its linkage to
multiple sclerosis: the usability and the hazard of a human
endogenous retrovirus. J Neurovirol 11, 232–235.
Dolei, A. (2006). Endogenous retroviruses and human disease. Expert
Rev Clin Immunol 2, 149–167.
ACKNOWLEDGEMENTS
Work was supported partly by grants from Fondazione Italiana Sclerosi
Multipla Onlus (grant no. 2005/R/11), Ministero Università e Ricerca
272
and Regione Autonoma Sardegna. We thank L. Poddighe for PBMC
RNA extraction. G. M. is a PhD fellow of the European Community.
The anti- HERV-W env 6A2B2 mAb was provided by bioMérieux.
Dolei, A., Serra, C., Mameli, G., Pugliatti, M., Sechi, S., Cirotto, M. C.,
Rosati, G. & Sotgiu, S. (2002). Multiple sclerosis-associated
retrovirus (MSRV) in Sardinian MS patients. Neurology 58, 471–473.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Journal of General Virology 88
MSRV/HERV-W and HHV-6 in multiple sclerosis
Donati, D., Akhyani, N., Fogdell-Hahn, A., Cermelli, C., CassianiIngoni, R., Vortmeyer, A., Heiss, J. D., Cogen, P., Gaillard, W. D. & other
authors (2003). Detection of human herpesvirus-6 in mesial temporal
lobe epilepsy surgical brain resections. Neurology 61, 1405–1411.
Mi, S., Lee, X., Li, X., Veldman, G. M., Finnerty, H., Racie, L., LaVallie, E.,
Tang, X. Y., Edouard, P. & other authors (2000). Syncytin is a
captive retroviral envelope protein involved in human placental
morphogenesis. Nature 403, 785–789.
Firouzi, R., Rolland, A., Michel, M., Jouvin-Marche, E., Hauw, J. J.,
Malcus-Vocanson, C., Lazarini, F., Gebuhrer, L., Seigneurin, J. M. &
other authors (2003). Multiple sclerosis-associated retrovirus
Moore, F. G. A. & Wolfson, C. (2002). Human herpes virus 6 and
particles cause T lymphocyte-dependent death with brain hemorrhage in humanized SCID mice model. J Neurovirol 9, 79–93.
human endogenous retroviruses in the placenta: an update. Placenta
25 (Suppl. 1), S16–S25.
Forsman, A., Yun, Z., Hu, L., Uzhameckis, D., Jern, P. & Blomberg, J.
(2005). Development of broadly targeted human endogenous
gammaretroviral pol-based real time PCRs quantitation of RNA
expression in human tissues. J Virol Methods 129, 16–30.
Fotheringham, J. & Jacobson, S. (2005). Human herpesvirus 6 and
multiple sclerosis: potential mechanisms for virus-induced disease.
Herpes 12, 4–9.
Garson, J. A., Tuke, P. W., Giraud, P., Paranhos-Baccala, G. &
Perron, H. (1998). Detection of virion-associated MSRV-RNA in
serum of patients with multiple sclerosis. Lancet 351, 33.
Garson, J., Créange, A., Dolei, A., Ferrante, P., Jouvin-Marche, E.,
Marche, P. N., Rieger, F., Ruprecht, K., Saresella, M. & other
authors (2005). MSRV, syncytin and the role of endogenous
retroviral proteins in demyelination. Mult Scler 11, 249–250.
Goodman, A. D., Mock, D. J., Powers, J. M., Baker, J. V. & Blumberg,
B. M. (2003). Human herpesvirus 6 genome and antigen in acute
multiple sclerosis lesions. J Infect Dis 187, 1365–1376.
Griscelli, F., Barrois, M., Chauvin, S., Lastere, S., Bellet, D. &
Bourhis, J. H. (2001). Quantification of human cytomegalovirus
DNA in bone marrow transplant recipients by real-time PCR. J Clin
Microbiol 39, 4362–4369.
Hollsberg, P., Kusk, M., Bech, E., Hansen, H. J., Jakobsen, J. &
Haahr, S. (2005). Presence of Epstein-Barr virus and human
herpesvirus 6B DNA in multiple sclerosis patients: associations
with disease activity. Acta Neurol Scand 112, 395–402.
Johnston, J. B., Silva, C., Holden, J., Warren, K. G., Clark, A. W. &
Power, C. (2001). Monocyte activation and differentiation augment
human endogenous retrovirus expression: implications for inflammatory brain diseases. Ann Neurol 50, 434–442.
Karlsson, H., Schröder, J., Bachmann, S., Bottmer, C. & Yolken,
R. H. (2004). HERV-W-related RNA detected in plasma from
individuals with recent-onset schizophrenia or schizoaffective
disorder. Mol Psychiatry 9, 12–13.
Komurian-Pradel,
F.,
Paranhos-Baccala,
G.,
Bedin,
F.,
Ounanian-Paraz, A., Sodoyer, M., Ott, C., Rajoharison, A., Garcia,
E., Mallet, F., Mandrand, B., Perron, H. & other authors (1999).
Molecular cloning and characterization of MSRV-related sequences
associated with retrovirus-like particles. Virology 260, 1–9.
Kwok, S. & Higuchi, R. (1989). Avoiding false positives with PCR.
Nature 339, 237–238.
Lafon, M., Jouvin-Marche, E., Marche, P. N. & Perron, H. (2002).
Human viral superantigens: to be or not to be transactivated? Trends
Immunol 23, 238–239.
multiple sclerosis. Acta Neurol Scand 106, 63–83.
Muir, A., Lever, A. & Moffett, A. (2004). Expression and functions of
Noseworthy, J. H., Lucchinetti, C., Rodriguez, M. & Weinshenker,
B. G. (2000). Multiple sclerosis. N Engl J Med 343, 938–952.
Nowak, J., Januszkiewicz, D., Pernak, M., Liwen, I., Zawada, M.,
Rembowska, J., Nowicka, K., Lewandowski, K., Hertmanowska, H.
& Wender, M. (2003). Multiple sclerosis-associated virus-related pol
sequences found both in multiple sclerosis and healthy donors are
more frequently expressed in multiple sclerosis patients. J Neurovirol
9, 112–117.
Opsahl, M. L. & Kennedy, P. G. (2005). Early and late HHV-6 gene
transcripts in multiple sclerosis lesions and normal appearing white
matter. Brain 128, 516–527.
Perron, H., Geny, C., Laurent, A., Mouriquand, C., Pellat, J., Perret, J.
& Seigneurin, J. M. (1989). Leptomeningeal cell line from multiple
sclerosis with reverse transcriptase activity and viral particles. Res
Virol 140, 551–561.
Perron, H., Suh, M., Lalande, B., Gratacap, B., Laurent, A., Stoebner, P.
& Seigneurin, J. M. (1993). Herpes simplex virus ICP0 and ICP4
immediate early proteins strongly enhance expression of a retrovirus
harboured by a leptomeningeal cell line from a patient with multiple
sclerosis. J Gen Virol 74, 65–72.
Perron, H., Firouzi, R., Tuke, P., Garson, J. A., Michel, M., Beseme, F.,
Bedin, F., Mallet, F., Marcel, E. & other authors (1997a). Cell
cultures and associated retroviruses in multiple sclerosis.
Collaborative Research Group on MS. Acta Neurol Scand Suppl 169,
22–31.
Perron, H., Garson, J. A., Bedin, F., Beseme, F., Paranhos-Baccala, G.,
Komurian-Pradel, F., Mallet, F., Tuke, P. W., Voisset, C. & other
authors (1997b). Molecular identification of a novel retrovirus
repeatedly isolated from patients with multiple sclerosis. The
Collaborative Research Group on Multiple Sclerosis. Proc Natl Acad
Sci U S A 94, 7583–7588.
Perron, H., Jouvin-Marche, E., Michel, M., Ounanian-Paraz, A.,
Camelo, S., Dumon, A., Jolivet-Reynaud, C., Marcel, F., Souillet, Y.
& other authors (2001). Multiple sclerosis retrovirus particles and
recombinant envelope trigger an abnormal immune response in
vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
Virology 287, 321–332.
Perron, H., Lazarini, F., Ruprecht, K., Pechoux-Longin, C., Seilhean, D.,
Sazdovitch, V., Creange, A., Battail-Poirot, N., Sibai, G. & other
authors (2005). Human endogenous retrovirus (HERV)-W ENV and
GAG proteins: physiological expression in human brain and
pathophysiological modulation in multiple sclerosis lesions.
J Neurovirol 11, 23–33.
Lolli, F., Mulinacci, B., Carotenuto, A., Bonetti, B., Sabatino, G.,
Mazzanti, B., D’Ursi, A. M., Novellino, E., Pazzagli, M. & other
authors (2005). An N-glucosylated peptide detecting disease-specific
Pfaffl, M. W. & Hageleit, M. (2001). Validities of mRNA quantifica-
autoantibodies, biomarkers of multiple sclerosis. Proc Natl Acad Sci
U S A 102, 10273–10278.
Rotola, A., Merlotti, I., Caniatti, L., Caselli, E., Granieri, E., Tola, M. R.,
Di Luca, D. & Cassai, E. (2004). Human herpesvirus 6 infects the
Menard, A., Amouri, R., Michel, M., Marcel, F., Brouillet, A.,
Belliveau, J., Geny, C., Deforges, L., Malcus-Vocanson, C. &
other authors (1997). Gliotoxicity, reverse transcriptase activity
central nervous system of multiple sclerosis patients in the early
stages of the disease. Mult Scler 10, 348–354.
and retroviral RNA in monocyte/macrophage culture supernatants
from patients with multiple sclerosis. FEBS Lett 413, 477–485.
http://vir.sgmjournals.org
tion using recombinant RNA and recombinant DNA external
calibration curves in real-time RT-PCR. Biotechnol Lett 23, 275–282.
Serra, C., Sotgiu, S., Mameli, G., Pugliatti, M., Rosati, G. & Dolei, A.
(2001). Multiple sclerosis and multiple sclerosis-associated retrovirus
in Sardinia. Neurol Sci 22, 171–173.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
273
G. Mameli and others
Serra, C., Mameli, G., Arru, G., Sotgiu, S., Rosati, G. & Dolei, A.
(2003). In vitro modulation of the multiple sclerosis (MS)-associated
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
retrovirus by cytokines:
J Neurovirol 9, 637–643.
pathogenesis.
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
Sotgiu, S., Pugliatti, M., Sanna, A., Sotgiu, A., Castiglia, P., Solinas, G.,
Dolei, A., Serra, C., Bonetti, B. & Rosati, G. (2002a). Multiple
Tuke, P. W., Hawke, S., Griffiths, P. D. & Clark, D. A. (2004).
implications
for
MS
sclerosis complexity in selected populations: the challenge of Sardinia,
insular Italy. Eur J Neurol 9, 329–341.
Sotgiu, S., Serra, C., Mameli, G., Pugliatti, M., Rosati, G., Arru, G. &
Dolei, A. (2002b). Multiple sclerosis-associated retrovirus and MS
prognosis: an observational study. Neurology 59, 1071–1073.
Sotgiu, S., Arru, G., Söderström, M., Mameli, G., Serra, C. & Dolei, A.
(2006). Multiple sclerosis-associated retrovirus and optic neuritis.
Mult Scler 12, 357–359.
Sotgiu, S., Arru, G., Mameli, G., Serra, C., Pugliatti, M., Rosati, G. &
Dolei, A. (2007). MSRV in early multiple sclerosis: a six-year follow-
up of a Sardinian cohort. Mult Scler (in press).
274
Distribution and quantification of human herpesvirus 6 in multiple
sclerosis and control brains. Mult Scler 10, 355–359.
Virtanen, J. O., Zabriskie, J. B., Siren, V., Friedman, J. E., Lyons, M. J.,
Edgar, M., Vaheri, A. & Koskiniemi, M. (2005). Co-localization of
human herpes virus 6 and tissue plasminogen activator in multiple
sclerosis brain tissue. Med Sci Monit 11, BR84–BR87.
Yi, J.-M., Kim, H.-M. & Kim, H.-S. (2004). Expression of the human
endogenous retrovirus HERV-W family in various human tissues
and cancer cells. J Gen Virol 85, 1203–1210.
Yoshikawa, T., Asano, Y., Akimoto, S., Ozaki, T., Iwasaki, T., Kurata, T.,
Goshima, F. & Nishiyama, Y. (2002). Latent infection of human
herpesvirus 6 in astrocytoma cell line and alteration of cytokine
synthesis. J Med Virol 66, 497–505.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 15 Jun 2017 08:48:53
Journal of General Virology 88