Journal of General Virology (1994), 75, 663-668. Printedin Great Britain
663
A single amino acid change in the E2 spike protein of a virulent strain of
Semliki Forest virus attenuates pathogenicity
Gwendoline M . G l a s g o w , 1 H e l e n M . Killen, 1 Peter Liljestr6m, 2 Brian J. S h e a h a n 3
and G r e g o r y J. A t k i n s 1.
1 Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland, 2 Department of Molecular
Biology, Karolinska Institute, S-141 57 Huddinge, Sweden and 3 Department of Veterinary Pathology,
Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dubl#1 4, Ireland
The virulent strain SFV4 of Semliki Forest virus (SFV),
produced from the infectious clone pSP6-SFV4, is lethal
after intranasal (i.n.) infection of adult mice and for
pregnant mice after intraperitoneal (i.p.) infection. In
contrast, the A7 strain of SFV is avirulent when given
i.n. to adult mice, but induces fetal death in pregnant
mice after i.p. infection. The nucleotide and deduced
amino acid sequences of part of the core and all of the
envelope region of A7-SFV were determined and
compared to those of SFV4. A7 differed from SFV4 at
80 nucleotides (nt) in the coding sequence, 15 of which
were associated with amino acid differences and seven of
which (two in the E2 protein and five in El) were nonconservative. The 3' non-coding sequence of A7 was
longer (415 nt) than that of SFV4 (263 nt) and a
divergent sequence of 181 nt was present adjacent to the
end of the E1 coding region. The effects on virulence of
two mutations in the E2 gene of SFV4, resulting in the
non-conservative amino acid substitutions present in
A7, were analysed. One mutation (mut 8729 a/c) resulted
in only slight attenuation, whereas the other (mut 8902
a/g) resulted in avirulence for pregnant mice. However,
mut 8902 a/g was lethal for the majority of developing
fetuses after i.p. infection of the mother.
Semliki Forest virus (SFV) is a member of the Alphavirus
genus of the family Togaviridae. In nature it infects
mosquitoes, small rodents and man (Mathiot et al.,
1990). It has been used extensively as a laboratory model
for the study of the molecular biology of alphavirus
multiplication (Schlesinger & Schlesinger, 1990) and
pathogenicity for the central nervous system (CNS;
Atkins et al., 1985; Peters & Dalrymple, 1990) and fetus
(Atkins et al., 1982; Mabruk et al., 1988, 1989; Milner &
Marshall, 1984). All SFV natural isolates are lethal when
administered to neonatal mice by any route. However, in
adult mice, differences in virulence between strains can
be distinguished according to the route of infection
(Bradish et al., 1971). The prototype strain (Henderson
et al., 1970), for which the sequence is known (Garoff et
al., 1980a, b; Takkinen, 1986), is avirulent when given
intraperitoneally (i.p. ; Morein et al., 1978 ; Snijders et al.,
1989, 1991). In comparison, the highly neurovirulent L10
strain is lethal when administered by any route (Bradish
et al., 1971), due to a lethal threshold of damage to
neurons in the CNS (Smithburn & Haddow, 1944).
Strains such as A7 (McIntosh et al., 1961) or M9 are
avirulent but induce demyelination by a mechanism that
involves the infection of oligodendrocytes (Atkins, 1983 ;
Atkins et al., 1990; Sheahan et al., 1983; Gates et al.,
1984, 1985; Fazakerley & Webb, 1987; Smyth et al.,
1990; Balluz et al., 1993). A7 is also lethal for developing
fetuses (Atkins et aI., 1982) and mutants derived from it,
such as ts22, are teratogenic (Hearne et al., 1987).
In an effort to define the molecular control of
pathogenicity for the CNS and fetus, we have utilized the
infectious clone pSP6-SFV4 of SFV (Liljestr6m et al.,
1991). Following transcription and electroporation into
BHK cells, this produces the SFV4 strain of virus which
is virulent when given intranasally (i.n.) to adult mice
(Glasgow et al., 1991). Our strategy is to introduce
sequence changes, present in the avirulent A7 strain of
SFV, into the SFV4 strain via the infectious clone, and to
measure changes in pathogenicity for the CNS and fetus.
Here we show that the envelope proteins of A7 have 15
amino acid changes compared to the SFV4 strain. Based
on the sequence results, we used site-specific mutagenesis
to introduce two of the changes in the E2 protein into the
SFV4 virus, and have shown that a single amino acid
change attenuates virulence.
The nucleotidesequencedata reported in this paper will appear in
the EMBL,GenBankand DDBJ nucleotidesequencedatabasesunder
the accessionnumberX74491.
0001-2061 © 1994 SGM
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Short communication
i
10
1 gaacgaatgt
81 ggtgggogac
161 aatogagoaa
241 oacgagggac
321 acagggagao
401 caagoacago
481 goaacgctga
561 ogaaaaaaac
641 oottgaogtg
721 atcgcgtact
801 cgaogggatg
881 acgoagaogg
961 acaatgggao
1041 ccgtgootgc
1121 aagagatcoo
1201 ocggacagga
1281 ctgtggaacc
1361 tgacagaooa
1441 atcoogttac
1521 gacactgcac
1601 gggtgacago
1681 aggatttggt
1761 ocogg~got
1841 ctgoggcccg
1921 tgcgcaccac
2001 ggagtttgog
2081 ottttttagt
2161 ~cgtataagg
2241 aa~octtaat
2321 agtgotc~ao
2401 tg~ttotgcg
2481 tgcttacaaa
2561 aogtgaa~gg
2641 gaaaa~aaga
2721 oggcgaaatc
2801 g~acggt~oa
2881 aaggotooat
2961 gaatttgoct
3041 eg~aatoctc
3121 tataaogtag
3201 cgoatoacot
3281 tagto~cata
3361 togggtggt~
3441 gtaoct~att
3521 gtaattttat
3601 ta~tataatc
3681 tataacaaag
3761 attaattggc
3841 tta
I
20
goatgaagat
aaagtcatga
gtatgacott
actataaotg
cgtggccggo
aotgtoggtg
ttactgacat
gcagaggcta
ccgaaacgga
gcgocgaatg
ctgaagattc
gcaogocatt
aottoatact
aggata~aat
ttgcaaoact
cgttgotatc
ggaaacgttg
taagaaatgg
cgttggacaa
ottcaoccag
ggoggtggaa
ctoaactcac
a~agoatccg
cagcaagtgt
gggagoatg~
gco~otgttg
gctactgagc
ot~aoattga
ttggaata~a
taaagagaag
actaagaaaa
gcccaoa~ag
agaocatgco
tagtogtgta
caaagoagaa
tgtao~ttaa
ttgg~tg~aa
gacagcgcct
ggatttoggc
ctactatgca
tcttttgtgg
tgogg~tag~
tggggg~ott
ttagoataca
tataactact
acttgaacta
cgcaaoaaga
aataattgga
I
30
tgaaaatgac
aaactgccca
gagtgtgocc
gaaacacggg
aoatttttga
gtoacotgga
gtgtgtcatt
ca~tacggat
aoaagacaco
oggagoaggg
agttctcggc
gagaacgccg
ggcaacgtgc
accateatga
tatcagcaaa
acagcaatct
g~actactag
oagttcaaot
tatoacatgt
atoatcccac
oggacoatao
oaotgaaggg
aggtcgccgg
ttgaococtt
agaoagtgtg
~ctgcatcct
ctcggggooa
aaggccagga
taacctgtga
oatgactatc
oaagoaaotc
oatogctgaa
gtcacgatag
oaaagacgaa
oggtggagag
acacagacao
aat~aaaaaa
ttactcgcat
ggagtcttga
ggaggccaca
tgtagctatg
oaoagtaacg
cgoaatcgg~
gggtacoaaa
tgaacagaaa
aaaaotggaa
ootgogoaat
agcttaaata
I
4o
tgtatcttcg
cgtgaaagga
agataeoagt
gocgttaagt
caaoaagggg
acaaagatat
gocaatgata
gctcgaggat
ggcgcagcgt
cactcgtgtc
aoaaattggc
tccggtoatc
ocaoogggtg
ccctcaaccg
caacagcgga
ggcaacgtaa
ttogga~atg
caccttttgt
agagttccaa
gct~ttttcc
ccgtaoeagt
aaa~ogcacg
gatgagctta
atgotttaao
gcagagacta
oat~atoa~g
ocg~aagagc
tatago~a~
gtaoaagacg
aatg~aaggt
agcgaggcgt
ggc~aaagtg
ggggta~t~a
gtgttcaatc
taatgacctg
cttcagggtt
aac~atgtoa
tgttgaggca
cattgaagta
gcaaaagtga
cagtgcoaag
tagtgtttoc
g~tattctgg
ttcttagott
actggaaaat
aacagaaaaa
tggccc~gta
agct~aatta
l
so
aagtcaaaca
gtcatcgaca
toacatgagg
acagcggagg
agggtagtcg
ggtgactaga
oattoccgtg
aacgtggata
gtcgcaaaac
atagcoccgt
atagataaga
tttgaaggta
aatttctaca
gtgggtagag
gaccgtggag
agatcacagt
acgatcaaca
aocgagagcc
tggogogoga
taocgcacac
ggaagggatg
9ctggccgca
otggcgttga
a~aaggagct
tggcctactt
tattgcct~a
ttacgaaoat
t~aotctgca
gt~gtcccgt
tta~a~aggc
acgtogatcg
agagttatgt
gttoatattc
aggaottccc
tacgcgaaca
caaatattgg
gggo~atgaa
coga~cat~a
caaaaccgac
agacag~agg
g~aacctgtt
cgacatgtog
tg~tagttgt
aattgacagt
agaaaaagtt
gttagggtaa
gtcogcttca
gacgaataat
I
60
tgaaggaaag
aogcggaoct
tcggatgctt
gaggttoact
ctatcgtcct
gtgaooocag
ottccagccc
ggccagggta
ttcaacgtgt
agcaattgaa
gtgacaatca
gccacctoog
ggtttogatc
aaaaatttac
gaaatogaca
aggaggaaag
cgtgtctaat
gacgaaccgg
accaactgtc
tgggcgagga
gagtaccact
t~agategta
tatcgatctt
gcagttccgt
gtgggaccaa
gaaacgtg~t
togacagtaa
gatgcaggtt
cgccgta~gt
gtgtaeeeat
atcggaogta
acggcaacgt
gggccgctgt
gcogtacgga
eggeactgaa
ctaaaggaaa
otgcg~ogtg
ctgac~tgac
aagaacgggg
taaggtgaac
oagcgtcgtg
ggoacogcac
ggtoa~ttgc
ataooaocat
agggtaggca
gcaotggcat
cggaaactcg
tggattttta
I
7o
gtoactgggt
ggoaaaaata
caaagtaoac
ataccgaaag
gggcggggoo
aggggtcoga
acgtgtgaac
ctacgacoto
ataaggctac
gogatcaggt
tgactaoacg
gagattgttt
caggacacta
aattagacoa
tgaatatgoa
aaggtaaaat
agageagtg~
otagaaaagg
atoca~ggca
o~ogcagtat
ggggaaacaa
eagtaotaot
ogogtogtgc
ggacgctggg
aaooaagagt
gtgttgctgt
tg~cgaacgt
gtogaaaoca
gaagtgotgc
t~atgtgggg
tg~aagcatg
aaaa~agaoc
catoggootg
tctgggcaac
gctggegcgc
aagggaoago
ggaaa~atoo
ttgcaoagtg
actgctctgt
ttacacttct
tgago~cccg
tatcatgggt
attggactco
cataattagc
atgttagttt
ataaooataa
ggggoaacto
tbttattttg
I
80
acgcctgoct
gctttoaaga
gcatgagaag
gagogggcaa
aaogagggct
agagtggtoo
cttgctgata
ottoaggcag
acgcccttae
oogaggctac
aagataaggt
cgtacatggc
gaaatgcggt
caotatggaa
gccagatacg
acaactgoac
oacgtctcag
caaagtccat
aaagagaagt
cacgaggaat
ogacccagtg
atgggattta
tacatgatgg
gatactotgo
tgttctggtt
aagagccttt
ggtggggttc
gccttgaacc
ggctcct~ag
tgggg~atat
atcaogoato
gtggatgttt
gacgccgttc
cagggcgctt
ccttcaoccg
~ctaaatacg
ctgt~tocat
gctacctgta
acactcaoa~
ccacggcaag
aaagaccaea
gcagaaaatc
gcagataata
oaagggtact
attatacctc
ctatataatt
atattgacac
caattggttt
80
160
240
320
400
480
560
640
720
800
880
960
1040
1120
1200
1280
1360
1440
1520
1600
1680
1760
1840
1920
2000
2080
2160
2240
2320
2400
2480
2560
2640
2720
2800
2880
2960
3040
3120
3200
3280
3360
3440
3520
3600
3680
3760
3840
3843
Fig. l. SequenceofcDNA correspondingtotheYportion oftheSFV A7genome. Nucleotidelcorrespondsto position 7744inthe
published sequenceofSFV (Garoffetal.,1980a, b).
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The A7 strain of SFV was grown from single plaque
isolates as previously described (Atkins, 1983; Atkins &
Sheahan, 1982). Purified virion RNA from A7 was
prepared by discontinuous density gradient centrifugation of the virus grown in BHK cells, proteinase K
(50 gg/ml) digestion, phenol-chloroform extraction and
ethanol precipitation (Atkins et al., 1974; V/i/in/inen &
Kfifirifiinem 1980). Reverse transcription was carried out
using the Pharmacia kit (Pharmacia P-L Biochemicals).
This generates cDNA with cohesive E c o R I ends and
internal NotI restriction sites at each end to facilitate
removal of cDNA clones from plasmid DNA. A7 cDNA
was inserted into a pGEM-3Z plasmid (Promega), which
had been previously restricted with EcoRI and dephosphorylated with bovine alkaline phosphatase (Boehringer Mannheim). Constructs were transformed into
Escherichia coli strain JM109 and positive clones were
initially selected using blue/white screening (Sambrook
et al., 1989). Further screening was carried out by
Southern blotting, using two DNA probes prepared
from plasmid pSP6-SFV4 (the SFV cDNA clone). DNA
fragments were purified using the Geneclean Kit (Bio 101)
and biotinylated using the BioNick labelling system
(Gibco BRL). Probe/clone hybrids were visualized on
Southern blots using the Gibco BRL DNA detection
system. DNA sequencing was carried out by primer
extension and the dideoxynucleotide chain termination
method, using both Klenow and reverse transcriptase
polymerases (Promega). Initial sequencing of the ends of
the viral inserts was carried out using primers to the SP6
and T7 promoters of the cloning vector. Subsequent
primers were 20-mer oligonucleotides, synthesized from
the published sequence of SFV (Garoff et al., 1980a, b).
Sequencing reactions were labelled with [35S]dATP (New
England Nuclear) before analysis by gel electrophoresis
and autoradiography. Oligonucleotide mutagenesis was
performed on ssDNA from M 13rap 18, carrying the SFV
structural genes (Kunkel et al., 1987; Liljestr6m et al.,
1991). Oligonucleotides used were 5' CCCGGTGGGCACGTTGCCAG and 5' CTCCACGGTCTCCGCTGTGG, and the nucleotide sequences of relevant regions of
DNA from phage isolates were determined. Fragments
containing single mutations were excised from the
replicative forms of the phage DNA using the AsuII site
at nucleotide (nt) 7781 of the viral sequence and the
plasmid SpeI site, and inserted into the pSP6-SFV4
clone. Newly constructed mutant cDNA clones were
again sequenced across inserted fragments.
Mice were infected with 10~ p.f.u, of virus, in 5 gl of
PBS for the i.n. route, in 50 lal of PBS for the intra
cerebral (i.c.) route and in 0-5 ml of PBS for the i.p.
route. Adult mice were 40 days old, and neonates 3 days
old. Pregnant mice were infected i.p. at day 8 of
pregnancy (Hearne et al., 1987). To determine the effect
665
Table 1. Amino acid substitutions o f the A 7 strain
compared to the S F V 4 strain*
Nucleotide
changes
rout
rout
mut
rout
mut
rout
mut
mut
mut
rout
mut
rout
mut
rout
mut
8291
8527
8729
8902
9053
9518
9530
9584
10057
10208
10547
10754
10820
10831
10982
t/c
g/a
a/c
a/g
a/g
t/c
t/c
t/c
g/t
g/a
t/c
t/c
c/a
a/g
g/a
A m i n o acid
substitutions
V
V
K
K
N
V
V
V
A
R
M
I
T
N
R
to
to
to
to
to
to
to
to
to
to
to
to
to
to
to
A
I
T
E
S
A
A
A
S
K
T
T
K
D
K
* N u m b e r s refer to nucleotide positions with reference to the
published sequence of SFV (Garoff et al., 1980a, b). Nucleotide
changes (SFV4/mutant) and amino acid substitutions (SFV4 to
mutant) are indicated.
of virus infection on fetal development, pregnant mice
were killed and fetuses examined at day 17 of pregnancy.
Two cDNA clones of the A7 strain were isolated and
shown to contain envelope protein gene sequences of 3.5
and 3.8 kb. Both clones lacked the viral poly(A) tail at
the 3' end of the genome, and lacked either 8 or 9 nt from
the end of the 3' non-coding sequence. The sequence of
the 3" region of the A7 genome, from position 7744 in the
C gene, to within 8 nt of the 3" end of the genome was
determined (a total of 3436 nt; Fig. 1), In the coding
region, the A7 sequence showed 80 nt changes compared
to the SFV4 sequence. Fifteen of these resulted in amino
acid changes, seven of which were non-conservative, two
in the E2 gene and five in E1 (Table 1). The 3' non-coding
region was 415 nt long for A7-SFV compared to 263 for
the SFV4 strain (Garoff et al., 1980b). A sequence of
234 nt at the 3' end of the genome was similar for A7 and
the SFV4 strains, but sequences between this region and
the end of the E1 coding region were divergent for the
two strains. One other feature of A7 was the presence of
two stop codons (ochre and amber) at the end of the
open reading frame, whereas only one (ochre) is present
for the SFV4 strain.
We also carried out a sequence analysis on the
structural protein region of the pSP6-SFV4 clone.
Although this clone was based on the published sequence
of the prototype strain of SFV (Garoff et al., 1980a, b)~
we found that it differed from the prototype sequence at
one position, i.e. at 8902 where a glutamic acid codon
had been changed to a lysine codon. Direct RNA
sequencing of the genomic RNA preparation originally
used as the template for cDNA synthesis in constructing
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666
Short communication
Table 2. Virulence characteristics o f S F V strains
SFVstrain
Murine
host*
Route of
No.
No.
infection infected dead
Mean time
of death
(days)
(±S.E.M.)
A7
Adult
Neonate
Pregnant
i.n.
i.c.
i.p.
14
l0
6
0
10
0~:
NA'~
2.0 (±0.0)
NA
SFV4
Adult
Neonate
Pregnant
i.n.
i.c.
i.p.
5
9
6
5
9
6
4.2 (_+0.2)
2-0 ( ± 0.0)
6-5 (_+ 0-2)
SFV4Adult
(mut 8729 a/c) Neonate
Pregnant
i.n.
i.c.
i.p.
14
8
6
11§
8
6
5.4 (_+0-2)
2'0 (±0"0)
6.3 (_+0.3)
SFV4Adult
(mut 8902 a/g) Neonate
Pregnant
i.n.
i.c.
i.p.
13
7
6
11§
7
0 I[
5.2 (_+0.3)
2.0 (+0.0)
NA
* Adult mice were 40 days old, neonates 3 days old and pregnant
mice were infected at day 8 of pregnancy.
t NA, Not applicable.
:~ None produced litters.
§ Mice surviving the initial infection were challenged 14 days later
with the virulent L10 strain of SFV; all survived.
1] Pregnant mice were killed at day 17 of pregnancy and fetuses
examined: two showed the presence of implantation sites only, whereas
four showed a mixture of necrotic (15) and normal (10) fetuses.
the SFV cDNA clone confirmed the presence of the same
substitution; the virus stock used for cDNA synthesis
had clearly mutated at some point. Thus both the A7
strain and the prototype strain originally sequenced had
glutamic acid at this position in the E2 protein, whereas
the SFV4 strain had lysine.
To analyse what effects the different A7 substitutions
might have on pathogenicity, we used site-specific
mutagenesis of the pSP6-SFV4 clone. Previous studies of
the molecular basis of pathogenicity of positive-strand
viruses have implicated the viral structural proteins. For
Theiler's virus, demyelination, persistence and virulence
have been mapped to the structural protein region of the
genome (Brahic et al., 1991 ; Pritchard et al., 1993). For
Sindbis virus, a togavirus like SFV, virulence for mice
has been associated with a site on the E2 protein close to
position 8902 (Polo & Johnston, 1991; Schoepp &
Johnston, 1993) and with a single amino acid change in
the E2 protein which controls binding to neural cells
(Tucker & Griffin, 1991). Because of these previous
results, our first approach was to analyse the nonconservative substitutions in the E2 gene. We first
changed the A to a C at position 8729 (mut 8729 a/c) of
the SFV4 cDNA, resulting in a lysine (K) to threonine
(T) amino acid substitution. Since the SFV4 and
prototype sequences differed non-conservatively, we also
changed the A at position 8902 to a G residue, resulting
in a lysine (K) to glutamic acid (E) substitution, i.e. an
amino acid reversion at this position of SFV4 to the
prototype. The SFV4 virus and both mutant variants
were tested by i.n. infection of adult mice, i.c. injection of
neonatal mice and i.p. injection of pregnant mice (Table
2). All three were virulent for adult and neonatal mice,
and SFV4 and SFV4-(mut 8729 a/c) were also virulent
for pregnant mice. In these respects the results obtained
for both mutants were similar to previous results
obtained for SFV4 (Glasgow et al., 1991), although both
mutants appeared to be slightly attenuated compared to
SFV4 when given i.n. to adult mice. However, SFV4(mut 8902 a/g) was avirulent for pregnant mice, thus
clearly differing from SFV4, and was lethal for most
developing fetuses, which is similar to A7, although A7
is lethal for all developing fetuses.
Of the two non-conservative amino acid changes in the
E2 protein, only one (designated rout 8902 a/g) affected
pathogenicity, as shown by site-specific mutagenesis of
the infectious clone. However, it differed from A7 in that
it was lethal for most adult mice when given i.n. The
results for the pathogenicity of SFV4-(mut 8902 a/g) are
similar to those for a previously described mutant,
SFV4-mut 64 (Glasgow et al., 199l). SFV4-mut 64 was
originally found during construction of the infectious
clone, and the mutation it contains was corrected to give
the pSP6-SFV4 plasmid. We previously described the
location of this mutation to be that of SFV4-(mut 8902
a/g), i.e. a change at amino acid 162 of the E2 protein to
give a glutamic acid for lysine substitution. Our results
now show that the mutation of SFV4-mut 64 is located
close to that of SFV4-(mut 8902 a/g), and results in a
valine for aspartic acid substitution at amino acid
position 168 of the E2 protein. Thus the lesions in SFV4mut 64 and SFV4-(mut 8902 a/g) are located only six
amino acids apart, and give similar phenotypes, probably
reflecting a common domain involved in virulence. The
SFV4-(mut 8902 a/g) change (as compared to SFV4) is
present in the original SFV sequence, and thus may at
least partially explain the avirulence of the prototype
strain. Although no detailed pathogenicity study has
been carried out using the prototype virus, it is clearly
attenuated since 107 p.f.u. (i.p.) constitutes a non-lethal
dose (Snijders et al., 1989, 1991), whereas 104p.f.u.
results in 50 % mortality for the SFV4 strain (Glasgow et
al., 1991). Although SFV4-(mut 8902 a/g) has some of
the characteristics of A7, there must be other attenuating
mutations or sequences in the A7 genome, since SFV4(mut 8902 a/g) also retains some of the virulence of
SFV4. The A7 strain of SFV is in fact an independent
avirutent isolate (McIntosh et at., 1961). Furthermore,
there is a divergent sequence present in the A7 3' noncoding region compared to SFV4. For Sindbis virus, it
has been shown that the 3' non-coding region controls
the host range for chick and mosquito cells (Kuhn et al.,
1990). One possibility for SFV is that this region of the
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genuine controls cell tropism for the CNS. This possibility and others are currently under investigation.
We thank Dorothy Mooney for excellent technical assistance, and
Steven Whitehead and colleagues for oligonucleotide synthesis. This
work was supported by the Wellcome Trust, the Multiple Sclerosis
Society of Ireland, and the Health Research Board to G. J. A./B. S. and
by the Swedish Medical Research Council to P.L.
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