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Human cytomegalovirus (HCMV) infection is considered to be an exacerbating factor in patients with ulcerative colitis (UC). However, the pathogenicity of HCMV in the exacerbation of UC remains unclear. The lack of a model mimicking UC with HCMV infection has posed a challenge for research into the pathogenic mechanism of HCMV in flare of UC. Therefore, the aim of our study was to establish a new mouse model of UC with HCMV infection.We established latent murine CMV (MCMV) infection in T-cell receptor α knockout (TCR-α KO) mice at an early age by adjustment of viral dose. Next, we performed immunohistochemical analysis in various organs of infected adult TCR-α KO mice to prove the correlation between MCMV infection and development of colitis. We then assessed colitis histologically and cytokine expression in the colon of infected and uninfected TCR-α KO mice. Finally, the types of MCMV-infected cells in the inflamed colon were examined by immunohistochemical analysis.MCMV antigen–positive cells reappeared predominantly in the inflamed colon of TCR-α KO mice. Severe colitis developed in the infected TCR-α KO mice compared with uninfected mice, and Th1/Th17 and Th2 responses were strongly induced. MCMV-infected cells were mainly perivascular stromal cells including pericytes, expressing platelet-derived growth factor receptor-beta (PDGFR-β) and CXC chemokine ligand 12 (CXCL12).In this study, we established, to our knowledge, the first mouse model of UC with HCMV infection. This model is an excellent tool for clarifying the detailed pathogenicity of HCMV in the exacerbation of UC and developing new treatment strategy for active UC with HCMV infection.