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 [8]. 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 [11]. 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 [8] 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. Results 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 [13]. 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 atrophy 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 [14]. Age-related brain atrophy in this strain is minimal and did not reach statistical significance in our published CMV Infection as Immunomodulator in an MS Model experiments [14]. 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 [15]. 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. Discussion 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 [7]. However, most studies have been unable to confirm this purported HCMV association with MS. Meanwhile, a potential disease PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32767