MCMV (K181+, salivary gland passaged stock [40]). 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 [14]. Premature animal loss due to co-infection was not observed during this study. Viral assays 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 homogenate. Disability measurement The rotarod assay (Rotamex rotarod, Columbus Instruments, Columbus, OH) was performed as a functional outcome measure (assessment of disability) every month, as previously published [38]. 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 [14]. A T2 weighted three dimensional RARE sequence was utilized for data References 1. Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG (2000) Multiple sclerosis. N Engl J Med 343: 938–952. 2. Zuvich RL, McCauley JL, Pericak-Vance MA, Haines JL (2009) Genetics and pathogenesis of multiple sclerosis. Semin Immunol 21: 328–333. 3. Handel AE, Giovannoni G, Ebers GC, Ramagopalan SV (2010) Environmental factors and their timing in adult-onset multiple sclerosis. Nat Rev Neurol 6: 156–166. 4. Ascherio A, Munger KL, Simon KC, Vitamin D, and multiple sclerosis (2010) Lancet Neurol 9: 599–612. 5. Ascherio A, Munger KL, Lennette ET, Spiegelman D, Hernan MA, et al. (2001) Epstein-Barr virus antibodies and risk of multiple sclerosis: a prospective study. JAMA 286: 3083–3088. 6. Barzilai O, Sherer Y, Ram M, Izhaky D, Anaya JM, et al. (2007) Epstein-Barr virus and cytomegalovirus in autoimmune diseases: are they truly notorious? A preliminary report. Ann N Y Acad Sci 1108: 567–577. 7. Wroblewska Z, Gilden D, Devlin M, Huang ES, Rorke LB, et al. (1979) Cytomegalovirus isolation from a chimpanzee with acute demyelinating disease after inoculation of multiple sclerosis brain cells. Infect Immun 25: 1008– 1015. 8. Zivadinov R, Nasuelli D, Tommasi MA, Serafin M, Bratina A, et al. (2006) Positivity of cytomegalovirus antibodies predicts a better clinical and radiological outcome in multiple sclerosis patients. Neurol Res 28: 262–269. 9. Alcami A, Koszinowski UH (2000) Viral mechanisms of immune evasion. Immunol Today 21: 447–455. 10. Mocarski ES, Jr. (2004) Immune escape and exploitation strategies of cytomegaloviruses: impact on and imitation of the major histocompatibility system. Cell Microbiol 6: 707–717. 11. Rodriguez M, Oleszak E, Leibowitz J (1987) Theiler’s murine encephalomyelitis: a model of demyelination and persistence of virus. Crit Rev Immunol 7: 325–365. 12. Denic A, Johnson AJ, Bieber AJ, Warrington AE, Rodriguez M, et al. (2011) The relevance of animal models in multiple sclerosis research. Pathophysiology: the official journal of the International Society for Pathophysiology/ISP 18: 21–29. CMV Infection as Immunomodulator in an MS Model 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 [14]. 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]. FACS analysis 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]. Statistical analysis Intergroup differences were analyzed statistically using standard statistical methods in JMP! (SAS Institute, Cary, NC) and SigmaPlot (Systat Software, Chicago, IL). Author Contributions 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. 13. Fields BN, Knipe DM, Howley PM, Griffin DE (2001) Fields virology. Philadelphia: Lippincott Williams & Wilkins. xix, 3087 p. 14. Pirko I, Johnson AJ, Chen Y, Lindquist DM, Lohrey AK, et al. Brain atrophy correlates with functional outcome in a murine model of multiple sclerosis. Neuroimage 54: 802–806. 15. Pirko I, Johnson AJ, Chen Y, Lindquist DM, Lohrey AK, et al. (2011) Brain atrophy correlates with functional outcome in a murine model of multiple sclerosis. Neuroimage 54: 802–806. 16. Bray PF, Bloomer LC, Salmon VC, Bagley MH, Larsen PD (1983) Epstein-Barr virus infection and antibody synthesis in patients with multiple sclerosis. Arch Neurol 40: 406–408. 17. Sanders VJ, Felisan S, Waddell A, Tourtellotte WW (1996) Detection of herpesviridae in postmortem multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol 2: 249–258. 18. Myhr KM, Riise T, Barrett-Connor E, Myrmel H, Vedeler C, et al. (1998) Altered antibody pattern to Epstein-Barr virus but not to other herpesviruses in multiple sclerosis: a population based case-control study from western Norway. J Neurol Neurosurg Psychiatry 64: 539–542. 19. Jilek S, Schluep M, Meylan P, Vingerhoets F, Guignard L, et al. (2008) Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis. Brain 131: 1712–1721. 20. Jaquiery E, Jilek S, Schluep M, Meylan P, Lysandropoulos A, et al. Intrathecal immune responses to EBV in early MS. Eur J Immunol 40: 878–887. 21. Benedict CA, Loewendorf A, Garcia Z, Blazar BR, Janssen EM (2008) Dendritic cell programming by cytomegalovirus stunts naive T cell responses via the PD- L1/PD-1 pathway. Journal of immunology 180: 4836–4847. 22. Kano Y, Shiohara T (2000) Current understanding of cytomegalovirus infection in immunocompetent individuals. J Dermatol Sci 22: 196–204. 23. Sweet C (1999) The pathogenicity of cytomegalovirus. FEMS Microbiol Rev 23: 457–482. 24. Mocarski ES, Jr. (2002) Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol 10: 332–339. 25. Rubin RH (1989) The indirect effects of cytomegalovirus infection on the outcome of organ transplantation. JAMA 261: 3607–3609. PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32767