Theiler's murine encephalomyelitis virus (TMEV) is a member of the Picornaviridae family and causes a virus strain dependent pathology in the central nervous system of mice. The GDVII strain induces an acute and mostly fatal encephalomyelitis. In the few mice that survive, the virus is cleared by the immune system. In contrast, infection with the DA strain leads to a persistent infection, marked by inflammation and demyelination that resembles multiple sclerosis. In the DA-induced disease, macrophages play a crucial role because they contribute to demyelination by the secretion of toxic mediators. Moreover, they represent the main viral reservoir, hereby also underlining their essential role in TMEV persistence. The mechanism of this persistence is not yet understood and tools to investigate it directly, without the complexity imposed by experimental animals, are largely missing. By studying TMEV infection of RAW264.7 macrophages, we found that the DA strain establishes a persistent infection in these cells, in contrast to the neurovirulent GDVII strain. Whereas the GDVII strain was cleared within 4 to 5 days post infection, DA virions were still present after 1 year of cell cultivation. This persistently DA-infected macrophage cell line, which we have called DRAW, provides a model to investigate the interactions between the cellular and viral factors influencing persistence and to screen for anti-TMEV agents.