CMV Infection Attenuates the Disease Course in a Murine
Model of Multiple Sclerosis
Istvan Pirko1*, Rhonda Cardin2, Yi Chen3, Anne K. Lohrey3, Diana M. Lindquist4, R. Scott Dunn4, Robert
Zivadinov5, Aaron J. Johnson3
1Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America, 2Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center,
Cincinnati, Ohio, United States of America, 3Department of Neurology and Immunology, Mayo Clinic, Rochester, Minnesota, United States of America, 4Imaging Research
Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 5Department of Neurology and Buffalo Neuroimaging Analysis Center,
University of Buffalo, Buffalo, New York, United States of America
Recent evidence in multiple sclerosis (MS) suggests that active CMV infection may result in more benign clinical disease. The
goal of this pilot study was to determine whether underlying murine CMV (MCMV) infection affects the course of the
Theiler’s murine encephalitis virus (TMEV) induced murine model of MS. A group of eight TMEV-infected mice were co-
infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two
weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome
measures included (1) monthly monitoring of disability via rotarod for 8 months; (2) in vivo MRI for brain atrophy studies
and (3) FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced
the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was
significantly improved starting 3 months post-infection and beyond (p#0.024). In addition, their brain atrophy was close to
30% reduced at 8 months, but this was only present as a trend due to low power (p=0.19). A significant reduction in the
proportion of brain infiltrating CD3+ cells was detected in this group (p=0.026), while the proportion of CD45+ Mac1+ cells
significantly increased (p=0.003). There was also a strong trend for a reduced proportion of CD4+ cells (p=0.17) while CD8
and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this
MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development
of demyelination and may be utilized for the development of novel therapeutic strategies.
Citation: Pirko I, Cardin R, Chen Y, Lohrey AK, Lindquist DM, et al. (2012) CMV Infection Attenuates the Disease Course in a Murine Model of Multiple
Sclerosis. PLoS ONE 7(2): e32767. doi:10.1371/journal.pone.0032767
Editor: Steven Jacobson, National Institutes of Health, United States of America
Received September 19, 2011; Accepted January 30, 2012; Published February 29, 2012
Copyright: ? 2012 Pirko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the National MS Society, by intramural funds at the University of Cincinnati’s Waddell Center for Multiple Sclerosis, and by the
National Institutes of Heatlh (NINDS R01 NS058698). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Pirko@mayo.edu
Multiple sclerosis (MS) is the most common inflammatory
demyelinating disease of the central nervous system (CNS). MS is
the leading cause of non-traumatic disability among young adults
in their most productive years . Although the exact cause of MS
remains elusive, it is widely accepted that the pathology of MS is
mediated by the immune system in genetically susceptible hosts
. In addition to genetic risk factors, which determine the
susceptibility and may influence disease severity, environmental
factors are also suspected to contribute as disease initiating events
. Infections, especially of viral etiology, have long been
suspected as environmental factors that may contribute to the
development of MS in addition to environmental variables
including vitamin-D deficiency . Several viruses have been
suspected as MS triggers. Currently, the most commonly studied
virus which appears to be associated with MS is Epstein-Barr
Virus (EBV), a member of the Herpesviridae family . EBV
establishes a persistent infection in B cells. Interestingly, EBV is
also suspected to play a role in the pathogenesis of several classic
autoimmune diseases, including polymyositis, SLE, anti-phospho-
lipid antibody syndrome, rheumatoid arthritis, pemphigus vul-
garis, giant cell arthritis, Wegener’s granulomatosis, and polyar-
teritis nodosa . A second herpes virus, human cytomegalovirus
(HCMV), has also been proposed as a potential MS trigger .
However, more recent studies with extensive case ascertainment
failed to demonstrate such an association . Among systemic
autoimmune conditions, elevated HCMV IgG titers were observed
only in the sera of SLE patients .
In a recent study analyzing the role of active HCMV infection
in MS cases, multiple analyses demonstrated a clear association
between antibody positivity against HCMV and better clinical and
MRI outcomes . These analyses indicated that patients positive
for antibodies against HCMV had significantly older age of disease
onset, lower lifetime relapse rate, higher brain parenchymal
fraction (BPF)on volumetric MRI, suggesting less brain atrophy.
HCMV-positive patients who had higher antibody titer presented
with lower T2 weighted lesion load and higher BPF compared to
patients with lower levels. Of note, this doesn’t mean that the
antibody itself would be responsible for the protective effect;
instead, it implies that recent active infection overall has a
protective role, via a mechanism that can’t be directly clarified in
PLoS ONE | www.plosone.org1 February 2012 | Volume 7 | Issue 2 | e32767
MS patients, but could be clarified via mechanistic studies in
animal models of the above phenomenon. The above was the first
study to suggest a protective role of HCMV infection in MS .
HCMV encodes multiple genes which serve to down modulate the
immune response during infection. These immune suppressive
aspects of HCMV infection could account for the protective effects
observed in HCMV-infected MS patients [9,10].
The goal of our study was to determine the extent murine CMV
(MCMV) infection exerts a similar protective effect in a mouse
model of MS. If protective, this model system could be analyzed
further to identify potential therapeutic exploitations of the
molecular mechanism(s) responsible for this effect. In the current
study, we utilized the TMEV infection based model of MS .
Mice of susceptible strains develop a demyelinating disease
characterized by clinical features of progressive myelopathy,
similarly to progressive forms of MS [11,12]. Since this MS model
itself is also based on a viral infection, our study can also be viewed
as a bi-pathogenic infection model. Based on the clinical
observations reported by Zivadinov et al  our hypothesis was
that in TMEV infected SJL/J mice, MCMV co-infection will
favorably modify the disease course similar to findings observed
clinically. Our main outcome measures were disability as assessed
by monthly rotarod performance, brain infiltrating lymphocyte
analysis by flow cytometry, and MRI based brain atrophy
measurements. All of these measures demonstrated a potential
protective effect of underlying MCMV infection in this MS model.
1. Preservation of motor function in chronic TMEV
infected animals pre-infected with MCMV
To determine the effect of underlying MCMV infection in the
TMEV model of MS, a total of four groups of mice were
compared. One group of mice received MCMV i.p. 2 weeks prior
to i.c. infection with TMEV. The other group received i.p. PBS
injection as a control instead of MCMV. We chose to infect the
MCMV-infected mice with TMEV at 2 weeks after MCMV
infection to allow sufficient time for a MCMV-specific immune
response to be developed and for multiple tissue sites to be infected
with MCMV . Two additional groups received TMEV
infection first, followed by MCMV infection or PBS. As shown
in Figure 1, there was a significant (p#0.024) protective effect of
MCMV pre-infection (MCMV/TMEV group) from the stand-
point of rotarod detectable functional disability, which first
became significant at 3 months post TMEV infection and
persisted beyond that. We also noticed the effects of ongoing
motor learning resulting in better than baseline performance in
these mice; however, this was not statistically significant (p$0.34).
A similar protective effect was not seen in the TMEV/MCMV
group, where MCMV infection was 2 weeks after TMEV infection
(p$0.44). Of note, MCMV infection appeared to be controlled at
8 months after infection since in the mice co-infected with TMEV
as MCMV replication in the salivary glands, a site for persistent
replicating virus, was not detected utilizing the MCMV assay as
described under ‘‘methods’’. In addition, no adverse effects, such
as weight loss and inactivity, were observed during the acute
infection phase for both the co-infected mice.
2. MRI results related to the development of brain
We analyzed brain atrophy in the treated mice. As reported by
us previously, brain atrophy is a standard feature of the TMEV
infected SJL/J mice . Age-related brain atrophy in this strain is
minimal and did not reach statistical significance in our published
experiments . We therefore elected to determine the extent
underlying MCMV infection reduced brain atrophy in TMEV
infected animals. Measuring ventricular volumes, we observed a
close to 30% reduction in brain atrophy in TMEV infected
animals with underlying MCMV infection (Figure 2); however,
while suggestive of a trend towards a protective effect, this
experiment was underpowered to detect a statistically significant
difference (p=0.19). The main reason for this was the relatively
high standard deviation of ventricular volume in the studied
groups of mice. Based on the observed standard deviation, we
would have needed 14 mice per group to demonstrate a significant
difference. It is important to note that the normal aging of SJL/J
mice doesn’t include the development of significant atrophy, as
demonstrated earlier . In addition, brain MRI metrics other
than atrophy were not considered in this model, as the majority of
demyelinating lesions are located in the spinal cord and not in the
brain. In vivo spinal cord imaging was not considered due to the
limited resources available for our study.
3. Brain infiltrating lymphocyte analysis by FACS
To determine whether the preserved motor ability observed in
mice infected with MCMV prior to TMEV infection was due to
altered brain infiltrating lymphocytes, we collected and quantified
the expansion of CD45+ brain infiltrating immune cells at 8
months post infection. As shown in Figure 3, MCMV pre-infection
of TMEV infected mice resulted in a significant reduction reduced
numbers of CD3+ cells as a percentage of brain infiltrating CD45+
cells in the brain (p=0.026). As part of this decrease in CD3+ cell
proportion, CD4+ cells exhibited a trend towards reduction in
TMEV infected mice pre-infected with MCMV (p=0.17).
Meanwhile, the proportion of CD45+ Mac1+ cells significantly
increased in these animals (p=0.003, Figure 3). Finally, the
proportions of B220+ or CD8+ cells did not change significantly in
TMEV infected animals (data not shown) pre-infected with
MCMV. In contrast, we did not observe statistically significant
changes in the proportions of CD3, CD4, CD8, Mac-1 or B220
positive cells infiltrating the brains of TMEV infected mice that
were subsequently infected with MCMV 2 weeks later. Overall,
these data suggest that pre-infection with MCMV reduces the
proportion of CD3+ T cell infiltration in the brain of mice
subsequently infected with TMEV. In the same animals,
underlying MCMV infection increases the proportion of Mac-1+
macrophage infiltration in chronic TMEV infected animals.
Overall, our findings imply a beneficial immunomodulatory
effect of MCMV infection in the TMEV infection MS model.
MCMV prior to TMEV infection may therefore contribute to our
understanding to the clinical observation that underlying CMV
infection contributes to protection from MS. The overall reduction
in the proportion of CD3+ cells and the observed increase in the
proportion of Mac-1+ macrophages may contribute to this effect.
The role and significance of HCMV infection from the
standpoint of MS development remains controversial. Similarly
to most putative viral causes of MS, one key issue is the almost
ubiquitous positivity of the average population to markers of these
infections. A potential causative role for HCMV was suggested
over 30 years ago based on primate experiments in which a strain
of CMV was isolated from the brain and lymph nodes of a
chimpanzee that developed paralysis after intracerebral inocula-
tion with brain cell cultures derived from an MS case .
However, most studies have been unable to confirm this purported
HCMV association with MS. Meanwhile, a potential disease
CMV Infection as Immunomodulator in an MS Model
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initiating role for EBV was suggested as far back as 1983 in a study
where the HCMV infection rate and HCMV complement fixing
antibody production was found to be lower in MS cases .
However, a study on post-mortem brain tissue using PCR based
detection techniques failed to find any statistically significant
association between MS and common viral infections including
HCMV and EBV . Meanwhile, a study in Norway found
elevated titers to EBV but not to HCMV in MS cases .
Recently, Ascherio reported a pathogenic role of EBV in MS
whereas no similar association was found regarding HCMV .
More recently, EBV-specific CD8+ T-cell responses were shown
to be decreased in patients with clinically isolated syndrome (CIS).
In contrast, there was no difference between categories for EBV-
specific CD4+ T cell, or for HCMV-specific CD4+ and CD8+ T-
cell responses . Intrathecal enrichment in EBV-, but not
HCMV-specific CD8+ CTL was also reported in early MS
patients by another group . EBV but not HCMV IgG
antibody indexes were also increased in the CSF in this study.
These studies overwhelmingly demonstrate that while EBV may
be an important ‘‘trigger’’ of MS development, HCMV doesn’t
contribute to MS pathogenesis.
A surprising finding about HCMV was its potential protective
role in MS, both from the standpoint of MRI and functional
outcome measures, suggesting that HCMV infection in MS
patients results in a beneficial modulation of the immune response
. This previous study demonstrated that recent HCMV
infection either by 1) primary infection or 2) secondary infection
(since humans can be infected with multiple CMV strains), or 3)
reactivation of latent virus leading to recent replication has
occurred in those patients, as reflected by the increased HCMV
antibody titers. Therefore, it is possible CMV-specific T cells were
activated or cytokine induction occurred, which has had an
immunomodulatory effect resulting in attenuated MS phenotype.
It is clear that both HCMV and MCMV infection affect the
responsiveness of T cells since CMV infected dendritic cells can
modulate naive and antigen specific T cell responses . There is
also evidence that HCMV may cause activation of immunomod-
ulating and immune evasion mechanisms [22,23] which could
alter the adaptive immune processes involved in the pathogenesis
of MS . The role of HCMV as an immunomodulating virus
has been recognized in solid organ transplantation where it
appears to contribute to the immunosuppressed state .
Figure 1. MCMV infection preserves motor function in SJL/J mice undergoing TMEV induced demyelinating syndrome. SJL/J mice
infected with MCMV two weeks prior to TMEV infection have significantly higher rotarod scores compared to controls that received PBS injection
instead of MCMV The intergroup difference first reaches significance at 3 months, and remains significant beyond that time point (p#0.024). The
figure also shows data acquired in mice infected with MCMV two weeks after TMEV infection; the data in those mice is virtually identical to our PBS/
TMEV control group. Error bars represent SD.
CMV Infection as Immunomodulator in an MS Model
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Infection of mice with MCMV reflects the infection of humans
with HCMV in many respects. MCMV infects numerous tissues
and cell types during the acute phase of infection and establishes a
lifelong persistent or latent infection similar to HCMV .
Similar to HCMV, MCMV encodes viral genes which function to
evade or alter the host immune response [9,10]. Some of these
appear to contribute to the observed lifelong viral persistence,
while others exploit immune cells that contribute to antiviral
immunity . Both HCMV and MCMV inhibit MHC class I
expression on infected cells [9,24,26], and MCMV has been
shown to impair IFN-gamma induced MHC class II-dependent
antigen presentation by macrophages [27,28]. HCMV was also
demonstrated to inhibit the induction of HLA class II antigens by
IFN-beta dependent and independent molecules including ICAM-
1 and VCAM-1, which are thought to be involved in MS
pathogenesis . In addition, CMV-infected endothelial cells
have the capability to induce IFN-beta production . Interferon
beta represents the most commonly used disease modifying agent
for relapsing forms of MS . Another possible mechanism to
explain the effects of prior MCMV infection on TMEV-mediated
MS development could result from modulation of the immune
response to TMEV infection itself. In mouse infection studies with
either MCMV or other viruses, prior infection of mice with one
virus influences the immune response to other heterologous
infections [31,32,33]. Thus, prior MCMV infection in the TMEV
model of MS could lead to attenuation of the MS-like disease as
shown in our studies by modulation of the immune response to
TMEV infection. In addition, cytokines released as part of an
antiviral immune response can activate the hypothalamo-hypo-
physeal axis, resulting adrenal glucocorticoid release, which in turn
provides strong negative feedback on the further synthesis and
release of cytokines, and exerts an overall protective effect from the
detrimental consequences of an overactive immune response
[34,35]. Lastly, as another potential, but at this stage purely
speculative explanation to our observations of increased propor-
tion of Mac-1+ cells, these cells may contribute to a more efficient
elimination of TMEV infected CNS cells, and as such led to a
better overall outcome. In addition, macrophages may exert
immunosuppressive effects on T-cells, as commonly demonstrated
in cancer models.[36,37]
Given that our study was designed as a pilot project paving the
way to future larger scale proposals, there are clear limitations to
our data. These include the relatively low number of mice per
group, which resulted in being underpowered from the standpoint
of demonstrating significant MRI-based differences. In addition,
due to the same limitation, only one time point was studied with
FACS, and we only studied brain and not spinal cord samples -
ideally both should be assessed given the prevalence of TMEV in
the spinal cord at late time points. We utilized the most commonly
studied immune cell markers in the FACS based studies; however,
additional immune subset markers, cytokine assay, microarray
studies would enable us to study this phenomenon in more details,
and all the above are planned in future extensions of this study.
The 2-week lag between MCMV and TMEV infections was
Figure 2. MRI results. Ventricular volumetry at 8 months post TMEV infection. Lower numbers represent less atrophy. The observed close to 30%
lower atrophy in the MCMEV/TMEV group only showed a trend (p=0.19) due to low statistical power, which was the consequence of the relatively
high standard deviation observed in these groups. Of note, ventricular volumetry of SJL/J mice undergoing normal aging does not demonstrate the
development of significant atrophy as demonstrated earlier .
CMV Infection as Immunomodulator in an MS Model
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chosen because MCMV virus titers peak in the salivary gland at 2
weeks and all other tissues are infected and some are even starting
to be cleared from the tissue, indicating virus-specific immune
response. However, it is possible that a different lag time may have
resulted in enhanced immunomodulatory effects. We also did not
demonstrate the effects of MCMV alone on the observed outcome
measures, including there was no control group where SJL/J mice
would be infected with MCMV alone. Histology time course
analysis was also not done, but given that we clearly documented
significant differences in motor performance, and motor perfor-
mance in this model is determined by the extent of both
demyelination and axonal loss, we anticipate that quantitative
measures of the aforementioned would also have demonstrated
In conclusion, in the studied chronic-progressive model of MS,
MCMV infection prior to demyelinating disease induction resulted
in an attenuated clinical phenotype. Overall, the mechanisms by
which human or mouse CMV effectively results in reduced disease
activity in MS or in the studied MS model remain unclear.
However, this study recapitulates in a rodent model the clinical
observation that underlying HCMV infection is protective in
human MS. We plan to study the observed phenomenon in
additional details in future extensions to this study, which will
address all the limitations of the current study, and include
additional time points and outcome measures. In our view, the
presented new model system will enable us to gain insights into the
beneficial immunomodulatory mechanism(s) associated with this
common viral infection, and may pave the way to novel
therapeutic strategies in MS.
Materials and Methods
Mice and Experimental Infection
The study was approved by the institutional committee for
animal care and use at the University of Cincinnati (approval
number 06-10-09-01). 4 week old female SJL/J mice were
purchased from the Jackson Laboratory (Bar Harbor, Maine).
Four groups of 8 mice were studied: MCMV inoculation either
preceded the infection with TMEV by 2 weeks, or was established
2 weeks after TMEV. We also used 2 control groups where PBS
was used as control for the MCMV inoculation, either 2 weeks
before or 2 weeks after infection. To induce the MS-like
demyelinating disease, all groups received TMEV infection by
intracranial (i.c.) injection of 105PFU of TMEV from the DAV
strain as published earlier [38,39]. Mice that received MCMV
were inoculated by intraperitoneal (i.p.) injection of 16105PFU
Figure 3. Flow cytometric analysis of brain infiltrating immune cells in 8 months TMEV infected mice that were either sham/PBS
injected, pre- or post-infected with MCMV. Shown are CD45+ cells that are: A.) CD3+, B.) CD4+, and C.) Mac1+, respectively. The observed
reduction in CD3+ cells compared to PBS controls was significant (p=0.026), and so was the increase in MAC1+ cells (p=0.003). The decrease in CD4+
T-cells demonstrated a trend (p=0.17).
CMV Infection as Immunomodulator in an MS Model
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MCMV (K181+, salivary gland passaged stock ). Matching
control groups received PBS instead of MCMV (PBS/TMEV and
TMEV/PBS groups). All mice were followed for a total of 8
months after TMEV infection. Our earlier observations suggested
that mice reach their peak disability by approximately 240 days
post infection . Premature animal loss due to co-infection was
not observed during this study.
At the end of the study, tissues were collected to determine the
effects of co-infection on normal levels of MCMV or TMEV
infection. At 8 months post infection, salivary glands were
collected to determine whether MCMV continued to persist or
replicate as a result of the TMEV co-infection. As previously
described, 10% salivary gland tissue homogenates (w/v) were
sonicated and plaque assays performed on NIH 3T3 cells [41,42].
Following incubation of the monolayers for 6 days under
carboxymethylcellulose-2X media, the cells were stained with
Giemsa and plaques enumerated by light microscopy. The limit of
detection for the plaque assay is ,10 PFU/ml of tissue
The rotarod assay (Rotamex rotarod, Columbus Instruments,
Columbus, OH) was performed as a functional outcome measure
(assessment of disability) every month, as previously published
. Mice were trained on the rotarod daily for one week prior to
infection to minimize effects of motor learning.
Brain atrophy measurements
MRI based brain volumetry was performed to assess brain
atrophy at the last time point (8 months post TMEV). For image
acquisition, a Bruker Biospec 7 Tesla horizontal bore small rodent
MR imaging system was used as described earlier . A T2
weighted three dimensional RARE sequence was utilized for data
acquisition (TR: 1500 ms, effective TE: 65 ms, RARE factor: 16,
isometric 125 micron resolution, total acquisition time ,40 min-
utes). We analyzed atrophy by performing volumetric measure-
ments of the ventricular enlargement as reported earlier .
Briefly, volumetric analysis using the 3D ROI tool was conducted
using the Analyze software package, developed by Mayo Clinic’s
Biomedical Imaging Resource [43,44].
Mice were euthanized and their brains were harvested at 8
months post infection, immediately following the MRI acquisition.
Brain-infiltrating lymphocytes were isolated from mouse brain
through collagenase digestion and a percoll gradient as previously
described [45,46]. Inflammatory cells isolated from the brains of
each mouse were stained with anti-CD4 PE (BD catalog
#553730), anti-CD8 PerCP (BD catalog #553036), anti-CD3
APC (BD catalog #553066), anti-CD45 PE-Cy7 (BD catalog
#552848), anti-B220 FITC (BD catalog #553087), and anti-Mac-
1 FITC (BD catalog #55557396) antibodies. Samples were then
washed twice with fluorescence-activated cell sorting buffer,
resuspended in cold phosphate-buffered saline, and fixed in 1%
paraformaldehyde. Samples were then analyzed on a BD LSRII
instrument (BD Biosciences) [47,48].
Intergroup differences were analyzed statistically using standard
statistical methods in JMP! (SAS Institute, Cary, NC) and
SigmaPlot (Systat Software, Chicago, IL).
Conceived and designed the experiments: IP RC RZ AJJ. Performed the
experiments: IP RC YC AKL DML RSD AJJ. Analyzed the data: IP RC
RZ AJJ. Wrote the paper: IP RC RZ AJJ.
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CMV Infection as Immunomodulator in an MS Model
PLoS ONE | www.plosone.org7 February 2012 | Volume 7 | Issue 2 | e32767