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Journal of Virology, October 1999, p. 7965-7971, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Viral Load and a Locus on Chromosome 11 Affect the
Late Clinical Disease Caused by Theiler's Virus
Stéphanie
Aubagnac,
Michel
Brahic,* and
Jean-François
Bureau
Unité des Virus Lents, CNRS URA 1930, Institut Pasteur, 75724 Paris Cedex 15, France
Received 22 April 1999/Accepted 30 June 1999
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ABSTRACT |
Theiler's virus causes a persistent infection and a demyelinating
disease of mice which is a model for multiple sclerosis. Susceptibility to viral persistence maps to several loci, including the
interferon gamma locus. Inactivating the gene coding for the interferon
gamma receptor makes 129/Sv mice susceptible to persistent infection
and clinical disease, whereas inactivating the interferon gamma gene
makes C57BL/6 mice susceptible to persistent infection but not to
clinical disease. This difference in phenotype is due to the difference
in genetic background. Clinical disease depends on high viral load and
Tmevd5, a locus on chromosome 11. These results have
consequences for the identification of viruses which might be
implicated in multiple sclerosis.
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INTRODUCTION |
The DA strain of Theiler's virus, a
picornavirus, is responsible for a chronic demyelinating disease in
genetically susceptible mice (12, 22). This natural
infection is one of the best models for multiple sclerosis. During the
first 2 weeks following intracranial inoculation, the virus causes an
encephalomyelitis regardless of the genetic background of the mouse.
Neurons of the brain and spinal cord are the main cells infected at
this stage. All mice recover, although some of them may present with
sequellae, such as flaccid paralysis of the hind legs (38).
In genetically susceptible strains of mice, such as the SJL/J strain,
this early disease is followed by a lifelong persistent infection of
the white matter of the spinal cord. At this stage, the virus infects
predominantly macrophage and microglial cells but also oligodendrocytes
and possibly astrocytes (1, 10, 30, 31, 33). Persistence is
associated with chronic inflammation and primary demyelination, which
may cause gait disorders and spastic paralysis, particularly when the
mice are inoculated with a high dose of virus (105 or
2 × 106 PFU). In contrast, genetically resistant
mouse strains, such as the C57BL/6 and 129/Sv strains, eliminate
the virus after early gray matter encephalomyelitis. Inflammation and
demyelination does not occur in these mice.
Persistence of infection and demyelination are under the control of
several host genes (3, 4, 37). The haplotype of the major
histocompatibility complex class I H-2D gene has a major effect on viral persistence (2, 6, 24, 31, 34).
H-2Db strains, such as C57BL/6 and 129/Sv, are
resistant, and their resistance is dominant. The SJL/J strain, however,
is more susceptible to viral persistence than predicted by its
H-2Ds haplotype (6). A locus on
chromosome 10, close to the Ifng locus, explains most of
the susceptibility of this strain (5). The role of the
interferon gamma pathway in the persistence of the infection was tested
by inoculating 129/Sv mice in which the gene coding for the
interferon gamma receptor had been inactivated (129/Sv
Ifngr
/ mice). These mice were highly
susceptible to persistent infection and presented with very severe
neurological symptoms (16). The role of this pathway can
also be tested by using C57BL/6
Ifng/ mice, which are now available.
They offer the possibility of testing the role of the cytokine
itself in the resistance of an H-2b mouse
strain. In the present article, we report that these mice were
persistently infected, but at a low level, and that they did not show
any clinical signs. We also show that most of the difference of
susceptibility between the 129/Sv Ifngr/
and the C57BL/6 Ifng/ strains is due to
the difference of genetic background. A complete genome scan of two
F2 crosses showed that clinically apparent disease results
from two independent factors: high viral load and a locus on chromosome
11, which we called Tmevd5 (for Theiler's murine
encephalomyelitis virus demyelination locus 5). The data indicate that
Tmevd5 modifies the risk of clinical disease in mice
infected at a high level.
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MATERIALS AND METHODS |
Animals.
C57BL/6 mice were purchased from Janvier (Le
Genest-St-Isle, France), and 129/Sv mice were purchased from the
Institut Pasteur animal facility. C57BL/6
Ifng/ mice were obtained from the
Jackson Laboratory (Bar Harbor, Maine). 129/Sv
Ifngr/ mice were provided by Michel
Aguet (Institut Suisse de Recherches Expérimentales sur le
Cancer, Lausanne, Switzerland). These mice have been described
previously (11, 19). F2
Ifng/ and F2
Ifngr/ mice (see Results for the
definition of these crosses) were bred at the Institut Pasteur animal
facility. The mice were genotyped for Ifng or
Ifngr by PCR amplification with DNA extracted from tail
biopsies. The following sets of primers were used to distinguish between Ifng/,
Ifng+/, and wild-type mice: the first
(5'-AGAAGTAAGTGGAAGGGCCCAGAAG-3' and
5'-AGGGAAACTGGGAGAGGAGAAATAT-3') set of primers amplified a
220-bp product from the Ifng gene, and the second
(5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3' and
5'-ATCGACAAGACCGGCTTCCATCCGA-3') set of primers amplified a
375-bp product from the 2-kb neomycin resistance gene inserted into
exon 2 of the Ifng gene. Amplification was performed
with a Gene Amp kit (Bio-Rad Laboratories, Gaithersburg, Md.) and a 9600 reactor (Perkin-Elmer Cetus, Norwalk, Conn.). After denaturation at 94°C for 2 min, 30 cycles of DNA amplification were performed under the following conditions for the first set of primers: 94°C for
40 s, 60°C for 40 s, and 72°C for 15 s. For the
second set of primers, the annealing temperature was 65°C instead of
60°C. The following primers were used to distinguish between
Ifngr/,
Ifngr+/, and wild-type mice:
5'-CCCATTTAGATCCTACATACGAAACATACGG-3' and 5'-TTTCTGTCATCATGGAAAGGAGGGATACAG-3'. These primers are
located in the Ifngr gene, upstream and downstream,
respectively, from the neomycin cassette. Amplification was carried out
under the following conditions: 94°C for 2 min followed by 40 cycles
at 94°C for 40 s, 55°C for 40 s, and 72°C for 2 min.
Viral inoculation.
The DA strain of Theiler's virus was
produced by transfection of BHK-21 cells with the pTMDA plasmid as
described elsewhere (26). Three- to 4-week-old anesthetized
mice were inoculated intracranially with 104 PFU of the DA
strain in 40 µl of phosphate buffered saline (PBS). All mice were
observed once or twice a week to record clinical signs and mortality.
They were sacrificed at 6, 21, or 45 days postinoculation (p.i.),
depending on the experiment.
Histological analysis.
Anesthetized mice were perfused
through the left ventricle with PBS followed by 4% paraformaldehyde in
PBS. The spinal cords were dissected out, postfixed, embedded in
paraffin, and sectioned as previously described (1).
Detection of Theiler's virus capsid antigen in the paraffin sections
was carried out with a primary rabbit hyperimmune anti-capsid serum, a
secondary biotinylated goat anti-rabbit immunoglobulin, and the ABC
peroxidase detection system (labelled streptavidin biotin [LSAB]
peroxidase; Dako). Slides were counterstained with Mayer hematoxylin.
Extraction of RNA from the spinal cord and quantification of
viral RNA.
The assay has been described in detail elsewhere
(6). Briefly, mice were anesthetized and perfused with 20 ml
of PBS. Their spinal cords were removed, and total RNA was extracted by
the procedure of Chomczynski and Sacchi (8). For each mouse,
series of fivefold dilutions, starting with 10 µg of total RNA, were dot blotted on Hybond C-extra filters (Amersham Corp., Arlington Heights, Ill.). The blots were hybridized with a
32P-labeled cDNA probe (specific activity, greater than
108 cpm/µg) specific for the 5' extremity of the
Theiler's virus genome. The hybridized filters were analyzed with a
phosphorimager. For each sample, the highest dilution which gave a
positive hybridization signal was used to estimate the viral RNA
content. The results were expressed as a dilution factor that ranged
from 1 to 625 or, to perform statistical analyses, as a score ranging
from 0 to 4 (see Fig. 1 and 3 for examples of the relationship between the dilution factor and the score). The data were normalized by two
procedures. First, a duplicate blot was hybridized with a 
-actin-specific probe to correct, if necessary, for variations in
the amount of total RNA. Second, reference samples from previous blots
were repeatedly analyzed to adjust for small variations of
hybridization efficiency from blot to blot. In most cases, quantification was performed by visual observation of a phosphorimager printout. For the F2 Ifng/
cross, quantification was also done by image analysis. Both methods gave essentially identical results.
Genetic analysis.
DNA was extracted from tail biopsies by
standard techniques (5). Mouse genotypes were determined
with polymorphic microsatellite markers. The sequences of the PCR
primers used are available online (13a). The primers were
synthesized at Research Genetics, Genset, or the Institut Pasteur
oligonucleotide facility. Amplifications were performed as previously
described (28). The optimal annealing temperature varied
from 50 to 55°C. The PCR products were visualized on 5% agarose gels
stained with ethidium bromide. Genetic maps were constructed with the
Mapmanager program (25). Depending on the F2
cross and the markers, 91 to 151 mice were used to calculate genetic
distances. Exclusion mapping and localization of susceptibility loci
were performed with the Mapmaker/QTL program (21), and the
Kosambi function was used to calculate genetic distances.
Statistical analysis.
The mean and standard error of the
mean (SEM) of the score of viral persistence were computed for the
three genotypes at each locus. Means were compared by using analysis of
variance from the Statview F-4.5 package. To account for deviation of
the persistence score from the Laplace-Gauss distribution, an empirical
significance level was obtained by a Monte Carlo method (data not
shown). The F distribution was evaluated in two simulations of 20,000 random replicates, each under the assumption of no linkage between the genotype and the phenotype. For the simulations, persistence scores were those observed in the experiment and genotypes were randomly assigned to members of the F2 cross. For each of the two
crosses, the simulated distribution was very close to the F
distribution of the table, and this distribution was used during the
remainder of the study. The linkage between clinical signs and each
locus was tested by a 
2 test. Dates of appearance of
clinical signs were studied as a function of genotypes with the Logrank
test of the nonparametric Kaplan-Meier analysis from the Statview F-4.5
package. Statistical criteria for the genetic analysis were those from
Lander and Kruglyak (20). Two levels of significance were
used: suggestive linkage, when the probability of linkage by chance was
less than 1.6 × 103 (decimal logarithm of the odds
[LOD] score, >2.8), and significant linkage, when the probability of
linkage by chance was less than 5.2 × 105 (LOD
score, >4.3).
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RESULTS |
C57BL/6 mice become susceptible to persistent infection with
Theiler's virus after inactivation of the Ifng
gene.
C57BL/6 Ifng/ and
wild-type C57BL/6 mice were inoculated intracranially with
104 PFU of the DA strain and observed for 45 days to record
clinical signs and mortality. Some mice were sacrificed at 6, 21, or 45 days p.i. Their brains and spinal cords were dissected out to quantify
viral persistence by a dot blot assay or to localize viral antigens by immunocytochemistry.
Six days p.i., the level of infection in the brain was lower in C57BL/6
Ifng/ mice than in wild-type mice (mean
value for B6Ifng/
[mB6Ifng
/] = 1.25 ± 0.2 [n = 12];
mB6 = 2.1 ± 0.4 [n = 11]), although the difference was not statistically
significant (NS) (P = 0.0524) (Fig.
1). Twenty-one days p.i., the levels of
viral RNA in the spinal cord were low and very similar in the two
groups (mB6Ifng/ = 0.2 ± 0.1 [n = 17];
mB6 = 0.4 ± 0.2 [n = 11]; NS) (Fig. 1). However, the locations of infected cells were
different in the two groups. Infected cells were found in the white
matter of the spinal cord, in the vicinity of the central canal, in
three of seven C57BL/6 Ifng/ mice
examined. In contrast, a small number of infected cells were found in
the gray matter of the spinal cord of two of nine C57BL/6 mice, close
to the central canal (data not shown). Forty-five days p.i., the level
of infection in the spinal cord was very low in the C57BL/6
control mice, as expected. In contrast, significant amounts of viral
RNA were present in the spinal cords of C57BL/6 Ifng/ mice (Fig. 1). The levels of
infection were significantly different in the two strains
(mB6Ifng/ = 1.3 ± 0.4 [n = 15]; mB6 = 0.1 ± 0.1 [n = 12]; P = 0.0115).
Immunocytochemistry did not detect viral antigens in the spinal cords
of three wild-type mice examined, although there was mild inflammation
in the gray matter (Fig. 2). In contrast,
infected cells were found in the white matter of the spinal cord in 8 of 11 C57BL/6 Ifng/ mice. Furthermore,
this infection was associated with inflammation such as perivascular
cuffs, meningitis, and diffuse parenchymal infiltration (Fig. 2). None
of the C57BL/6 wild-type and C57BL/6 Ifng/ mice showed clinical signs or died
during this study.
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FIG. 1.
Amount of viral RNA in the central nervous system at
different times p.i. The amount of viral RNA was measured in the brain
on day 6 p.i. and in the spinal cord on days 21 and 45 p.i.
This amount is expressed as the highest RNA dilution which gave a
hybridization signal (left ordinate) or the score of viral persistence
(right ordinate). n, number of animals per group. *,
P < 0.05. The error bars indicate the SEM.
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FIG. 2.
Longitudinal sections of spinal cord from a C57BL/6
Ifng/ mouse (A) and a wild-type C57BL/6
mouse (B) with Theiler's virus 45 days p.i. The arrows indicate
infected cells detected by immunocytochemistry. Magnification, ×200
(A) and ×320 (B).
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In a previous work, we reported that 129/Sv
Ifngr/ mice were highly susceptible to
persistent infection with Theiler's virus
and died from extensive
white matter disease before 45 days p.i.
(
16). We
repeated these experiments to compare C57BL/6
Ifng/ and 129/Sv
Ifngr/ mice under the same conditions.
Forty-five days p.i., the level
of viral RNA was significantly lower in
C57BL/6
Ifng/ mice than in 129/Sv
Ifngr/ mice
(
mB6Ifng/ = 1.3 ± 0.4
[
n = 15];
m129Ifngr/ = 3.8 ±
0.1 [
n = 9];
P < 0.0001)
(Fig.
3). At that time, seven of
nine
129/Sv
Ifngr/ mice had developed
hind-leg paralysis whereas no symptoms were
observed in 26 C57BL/6
Ifng/ mice (Table
1). No mortality was observed in either
group of
knockout mice.
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FIG. 3.
Amount of viral RNA 45 days p.i. in the spinal cord of
the two F2 crosses and their parental strains. The amount
of viral RNA is expressed as the highest RNA dilution (+ SEM) which
gave a hybridization signal (left ordinate) or the score of viral
persistence (right ordinate). n, number of animals per
group. *, P < 0.05; **, P < 0.01; ***,
P < 0.001.
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Modifier genes controlling virus load and clinical signs explain
differences between C57BL/6 Ifng/ and
129/Sv Ifngr/ mice.
As discussed
above, C57BL/6 Ifng/ mice are less
susceptible to persistent infection with Theiler's virus and to the
associated clinical symptoms than 129/Sv
Ifngr/ mice. This could be due to the
fact that the gene which is inactivated codes for the cytokine in the
former strain whereas it codes for the cytokine receptor in the latter.
Alternatively, it could be due to the difference in genetic background
between the two strains. Because null mutants with the same
genetic background were not available, we tested these hypotheses
by comparing two F2 crosses (C57BL/6
Ifng/ × 129/Sv) F2
Ifng/ and (129/Sv
Ifngr/ × C57BL/6) F2
Ifngr/, for level of viral persistence
and appearance of symptoms. (For the sake of simplicity, these crosses
will be designated F2 Ifng/
and F2 Ifngr/, respectively,
in the rest of this article). If the difference in susceptibility were
due to the functional differences between interferon gamma and its
receptor, the F2 Ifng/ and
C57BL/6 Ifng/ mice should have the same
phenotype and the F2 Ifngr/ and 129/Sv
Ifngr/ mice should also have the same
phenotype. If, on the other hand, the difference in susceptibility were
due to the difference in genetic background, both crosses should have
similar phenotypes, with large variations between animals.
One hundred and seventeen F
2
Ifng/ and 151 F
2
Ifngr/ animals were inoculated and
monitored for clinical symptoms. After 45 days,
the level of viral
persistence was measured for each mouse. As
shown in Fig.
3, both
groups of F
2 mice were infected at a significantly
higher
level than the C57BL/6
Ifng/ strain
(
P = 0.0405 and
P = 0.0014,
respectively, for F
2 Ifng/
and F
2 Ifngr/) and at a
significantly lower level than the 129/Sv
Ifngr/ strain (
P < 0.0001 and
P = 0.0077, respectively, for
F
2 Ifng/ and F
2
Ifngr/). In contrast to the C57BL/6
Ifng/ mice, which remained asymptomatic,
14.5 and 26% of the F
2
Ifng/ and F
2
Ifngr/ mice, respectively, showed
paralysis. Mortality was 2.5 and 4%,
respectively (Table
1). The
incidence of clinical disease was
lower for both F
2 crosses
than for the 129/Sv
Ifngr/ strain
(78%). Taken together, these results imply that the difference
in
susceptibility between the C57BL/6
Ifng/
and the 129/Sv
Ifngr/ mice is due mainly
to the difference in genetic background. We
cannot rule out a
contribution of the difference in mutation,
since the F
2
Ifngr/ mice were infected at a slightly,
but significantly, higher level
than F
2
Ifng/ mice (
P = 0.0039)
and showed more clinical symptoms (
P = 0.0241).
Only
males contributed to the difference in susceptibility between
the
F
2 crosses
(
mF2Ifngr/male = 2.7 ± 0.1 [
n = 81];
mF2Ifng/male = 2.0 ± 0.20 [
n = 57];
P = 0.0008;
mF2Ifngr/female = 2.2 ± 0.2 [
n = 53];
mF2Ifng/female
= 2.1 ± 0.2 [
n = 53];
NS).
Clinical symptoms appeared at the same time in the two groups
of F
2 mice (mean time of appearance, 37 days p.i.).
Interestingly,
in both groups, clinical signs were associated with a
high level
of persistent infection: 42 of 46 mice with paralysis had a
persistence
score higher than 3 (Fig.
4).
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FIG. 4.
Amount of viral RNA at 45 days p.i. in the spinal cords
of asymptomatic (left) and symptomatic (right) mice.
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Localization of loci controlling viral persistence.
The 145 mice of the F2 Ifngr/ cross
and the 111 mice of the F2
Ifng/ cross were genotyped in an attempt
to map loci controlling viral persistence. The entire genome was
screened with 99 and 108 microsatellite markers, respectively. Two to
eight loci were analyzed for each chromosome, depending on the size of
the chromosome and on the presence or absence of a putative linkage.
The distance between adjacent loci varied from 1 centi-Morgan (cM) to
27 cM. No marker was linked with viral persistence, even at the
suggestive-linkage level, in either cross. However, in the
F2 Ifng/ cross, weak linkage
with viral persistence was observed on chromosome 19, close to
D19Mit123 (P = 0.0028), on chromosome 15 between D15Mit154 (P = 0.0059) and D15Mit156
(P = 0.0043), and on chromosome 12 between
D12Mit218 (P = 0.0029) and D12Mit106
(P = 0.0094). These results were not confirmed with the
F2 Ifngr/ cross.
Localization of loci controlling clinical disease.
The number
of clinically affected mice (n = 39) was large
enough in the F2
Ifngr/ cross to warrant looking for
linkage with polymorphic markers. This was not the case for the
F2 Ifng/ cross (n = 17). The F2 Ifngr/
mice were separated into two groups, affected and nonaffected, regardless of the time of appearance of the symptoms. Linkage was
tested at each locus by comparing the numbers of affected and
nonaffected mice as a function of genotype with a 2
test. There was significant linkage with clinical disease on chromosome
11 for marker D11Mit179 (P < 0.0001; LOD
score = 4.8) and suggestive linkage with the two flanking markers,
D11Mit4 and D11Mit99 (P = 0.0008, LOD
score = 3.1, and P = 0.0012, LOD score = 2.9, respectively) (Table 2). In the
nonaffected group, alleles segregated randomly. Susceptibility to
clinical disease was conferred by the C57BL/6 allele at the
D11Mit179 locus. Indeed, among the 39 affected mice, 26 were
homozygous for the C57BL/6 allelic form, 5 were homozygous for the
129/Sv allelic form, and 8 were heterozygous. This result was confirmed
with the Logrank test of the nonparametric Kaplan-Meier survival
analysis (2 = 24.468 [2 df]; P < 0.0001).
The results reported in a previous section of this paper suggested the
existence of a relationship between clinical symptoms
and a high level
of viral persistence. To examine the relationship
between clinical
signs, viral persistence, and genotype at the
D11Mit179
locus, the amounts of viral RNA in the nonaffected and
affected mice
were compared as a function of genotype (Fig.
5).
Clinical signs were associated with a
high level of persistent
infection regardless of the genotype. However,
when one compares
mice with high viral loads (with scores equal or
superior to 3.5),
77% of the C57BL/6 homozygous mice were affected as
opposed to
30% of the heterozygous mice and 36.5% of the 129/Sv
homozygous
mice (Fig.
5). Interestingly, there was only weak linkage
between
viral persistence and the
D11Mit179 marker
(
P = 0.0167). In conclusion,
our results strongly
suggest the presence on chromosome 11 of
a locus, which we named
Tmevd5, which controls the severity of
clinical disease but
not the viral load.
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FIG. 5.
Amount of viral RNA 45 days p.i. in the spinal cords of
asymptomatic (left-hand graphs) and symptomatic (right-hand graphs)
mice as a function of genotype at the D11Mit179 locus. For
each group of animals, the absolute number and the percentage of mice
with a score of viral persistence equal or superior to 3.5 are given.
These animals are indicated by hatched and solid boxes, respectively.
Six asymptomatic mice died before 45 days p.i. (five were C57BL/6
homozygous, and one was 129/Sv homozygous for the D11Mit179
marker).
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In the F
2 Ifngr/ cross,
males had 2.2 times more viral RNA in their spinal cords than
females. Therefore, we decided to study
susceptibility to clinical
signs in males and females separately.
When doing so, two markers,
D19Mit65 and
D19Mit119, had suggestive
linkage with disease only in females (
P = 0.0011 and
P = 0.0013,
respectively) (Table
3). The segregation of alleles in males
and in nonaffected females was random, but in affected females
there
was a deficit in heterozygous animals. Therefore, there
might be
another locus, on chromosome 19, which controls clinical
symptoms.
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DISCUSSION |
In a previous work, we used a cross between the SJL/J and B10.S
strains to identify a locus, close to Ifng, which
controls the persistence of Theiler's virus in the spinal cord
(5). Because of the antiviral and immunoregulatory
properties of interferon gamma, the Ifng gene was a good
candidate for the control of viral persistence. Studies with 129/Sv
mice in which the gene coding for the interferon gamma receptor had
been inactivated demonstrated the role of the interferon gamma pathway
in resistance to persistent infection (16, 35). The recent
development of a C57BL/6 strain whose gene coding for interferon gamma
has been inactivated allowed us to test the role of the cytokine
directly. Unexpectedly, these mice were less susceptible to viral
persistence and to clinical signs than the 129/Sv
Ifngr/ mice. The difference in
susceptibility between the 129/Sv Ifngr/
and C57BL/6 Ifng/ strains could have
been due to the functional differences existing between the cytokine
and its receptor or to the difference in genetic background between the
two mouse strains. By comparing the phenotypes of the F2
Ifng/ and the F2
Ifngr/ crosses (see Results for the
definition of these crosses), we showed that 80% of the difference in
susceptibility to viral persistence was due to the difference in
genetic background. Since there is a small, but statistically
significant, difference in viral load between the F2
Ifng/ and F2
Ifngr/ lines (Fig. 3), part of the
difference in susceptibility between the parental strains might be due
to the fact that the inactivated gene was different for each of them.
However, we cannot rule out the possibility that some 129/Sv
susceptibility loci were still present in the C57BL/6
Ifng/ strain, since it was obtained from
a 129/Sv ES cell line after only 10 backcrosses. 129/Sv
Ifng/ mice have been described by Cantin
et al. since this article was submitted for publication (7).
They will be invaluable to compare the roles of gamma interferon and
its receptor in the pathogenesis of this model disease.
The comparison of the phenotypes of the parental strains and of the two
F2 crosses showed that the level of viral persistence was
genetically controlled (Fig. 3). In spite of this, we did not find a
locus linked to viral persistence when a complete genome scan of both
crosses was performed. This is most likely because the number of loci
involved is too high to allow the detection of linkages with a cross of
approximately 100 animals. It has been reported that the C57BL/6 strain
is more resistant to demyelination than other
H-2Db strains (32). The existence of
a large number of non-H-2 resistance genes in the C57BL/6
strain might put limits on the use of null-mutant mice with this
background to study the pathogenesis of Theiler's virus infection. The
phenotypes of the mutant mice might be less pronounced than on another
resistant background, and also more difficult to interpret.
The main conclusion of the present work is that the appearance of
symptoms in this model disease depends on at least two independent factors: high viral load and a locus on chromosome 11 which we named
Tmevd5. In previous publications, we noted that the mouse strains with high persistent viral load, as measured with a dot blot
assay, were usually the strains identified by other groups as
susceptible to clinical disease (6, 27). In the present study we confirmed the association between high viral load and clinical
disease among mice from two different F2 crosses (Fig. 4
and 5). However, others who measured viral load with an infectivity assay did not find a correlation between viral persistence and clinical
disease (9, 23, 31). The reason for this discrepancy is not
clear at present. It could be due in part to trivial technical difficulties with the infectivity assay (the DA virus remains extremely
membrane associated in tissue extracts).
The Tmevd5 locus is significantly linked to clinical disease
but not, or only weakly, to viral persistence. This locus could act in
two different ways: (i) it could change the threshold of viral load
required for the appearance of symptoms, and (ii) it could change the
likelihood of appearance of symptoms for mice with a given viral load.
The data shown in Fig. 5 clearly favor the second possibility: all
symptomatic mice have a large viral load, regardless of genotype.
However, the fraction of animals with a large viral load which are
symptomatic is much higher when the animals are C57BL/6 homozygous at
the Tmevd5 locus. The characterization of the
Tmevd5 locus is now an important step in the study of the pathogenesis of this model disease. Interestingly, Idd4, a
locus involved in the insulin-dependent diabetes of the nonobese
diabetic mouse, is located in the same region (39). The
Tmevd5 C57BL/6 allele confers susceptibility, although the
C57BL/6 strain is resistant. Although this may seem surprising at
first, similar results have been reported with other polygenic diseases
(13, 17, 29). It is likely that, in the C57BL/6 strain, the
effect of a susceptible allele at one locus is overcome by the effects of resistant alleles at other loci. As shown by our work, the C57BL/6
allele at Tmevd5 affects clinical disease only in mice with
a high viral burden. The level of viral infection was probably too low
to observe the effect of this locus in the C57BL/6
Ifng/ mice.
Our findings may have implications for the study of diseases of humans.
Persistent viral infections may be asymptomatic or associated with
severe diseases in humans. Infection by human T-cell leukemia
virus type I is a case in point. With other viruses, such as
human immunodeficiency virus, the duration of the asymptomatic phase
can be highly variable from individual to individual. It is usually
assumed that, in these situations, the main determinant of clinical
disease is high viral burden. Our work with Theiler's virus shows that
the situation might be more complex and that host genetic factors can
influence the occurrence of clinical disease in individuals with the
same high viral load.
Multiple sclerosis is a multifactorial disease in which environmental,
presumably infectious, and genetic factors (14, 15, 18, 36)
interact to cause white matter damage. In mice, several common viruses,
including Theiler's virus and strains of coronaviruses, cause diseases
which resemble multiple sclerosis in genetically susceptible inbred
strains. By analogy with the murine models, one has to consider the
possibility that multiple sclerosis results from infections by common
human viruses in individuals with a specific genetic background which
renders them susceptible to demyelination by a particular virus. If
such were the case, the epidemiological studies performed so far to
test the viral hypothesis of multiple sclerosis would be unable to
establish a causal relationship between a given virus and the disease.
In conclusion, high viral load is necessary but not sufficient to cause
clinical disease in mice persistently infected with Theiler's virus.
The Tmevd5 locus on chromosome 11 modifies the risk of
disease in mice with a high viral burden. These results might have
important implications for the study of multiple sclerosis and other
multifactorial human diseases.
| |
ACKNOWLEDGMENTS |
We thank F. Bihl for helpful discussion, J.-L. Guénet, G. Millon, X. Montagutelli, and S. Wain-Hobson for critical reading of the
manuscript, and M. Gau for secretarial aid.
This study was supported by grants from the Institut Pasteur Fondation,
the Centre National de la Recherche Scientifique, the National Multiple
Sclerosis Society USA, the European Community Human Capital and
Mobility Program, and the Association pour la Recherche sur la
Sclérose en Plaques. S.A. is the recipient of a scholarship from
the Ministère de l'Education Nationale, de l'Enseignement
Supérieur et de la Recherche.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité des
Virus Lents, CNRS URA 1930, Institut Pasteur, 28, rue du Dr. Roux,
75724 Paris Cedex 15, France. Phone: 33 (0)1 45 68 87 70. Fax: 33 (0)1 40 61 31 67. E-mail: mbrahic{at}pasteur.fr.
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Journal of Virology, October 1999, p. 7965-7971, Vol. 73, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
