Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.