Prolonged gray matter disease without demyelination caused by Theiler's murine encephalomyelitis virus with a mutation in VP2 puff B

Abstract

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.

FIG. 1

(a) Ribbon representation of TMEV constructs. Shown in blue and purple are DA strain VP1 and VP2, respectively. DA strain VP1 loop I and VP2 puff A are colored dark gray at top center in the rear plane (not labeled). DA strain VP1 loop II and VP2 puff B are shown in red and yellow, respectively. GDVII strain VP1 loop II and VP2 puff B are shown in white and orange, respectively. (b) Model of the expected differences in TMEV constructs. Loop regions are shown for the superimposition of DA and GDVII strains. Panel a is a large view, while panel b is a detail view.

FIG. 2

One-step growth curve of TMEV mutants. We infected BHK-21 cells with wild-type pDA, DApB and DApBL2M viruses at an MOI of 5. Infected cells with supernatants were harvested at the indicated time points, and the virus titers were determined by a plaque assay. We could detect comparable amounts of viruses between the groups. Results are representative of two independent experiments.

FIG. 3

Clinical signs in TMEV infection. We injected SJL/J mice with pDA, DApB, or DApBL2M virus intracerebrally and observed weight change (a) and righting reflex (b). In the first 2 weeks after infection, the acute phase, all groups showed weight loss and a mild impaired righting reflex. During the chronic phase, more than 1 month after infection, pDA and DApB virus infected mice showed less weight gain than DApBL2M virus-infected mice (weight gains on day 114: pDA, 4.5 ± 0.3 g; DApB, 4.6 ± 0.3 g; DApBL2M, 6.0 ± 0.5; ∗P < 0.05 compared with pDA, analysis of variance). The righting reflex abnormality was less severe in DApB and DApBL2M virus-infected mice than in pDA virus-infected mice (righting reflex scores on day 114: pDA, 2.1 ± 0.1; DApB, 1.6 ± 0.1; DApBL2M, 1.3 ± 0.2; ∗∗, P < 0.01). Each experimental group consisted of 5 to 16 mice. Results are representative of two independent experiments.

FIG. 4

Neuropathology during the acute phase of TMEV infection. Mice were sacrificed 1 week after infection with pDA (a, b, and g), DApB (c and d), or DApBL2M (e, f, and h) virus. In all groups, mice developed similar inflammatory responses in the hippocampal fissure (a, c, and e, large arrow). More pyknotic neurons in the pyramidal cell layer (a, c, and e, arrowheads) of the hippocampus in DApB (c) and DApBL2M (e) virus-infected mice were observed. We detected more TMEV antigen-positive cells (b, d, and f, small arrows) in pDA virus-infected mice (b) than in DApB (d, inset) or DApBL2M (f, inset) virus-infected mice. Although the cerebellum was normal in pDA (g) or DApB virus-infected mice, DApBL2M virus-infected mice showed MNC inflammation (h, large arrows) in the cerebellum. (a, c, e, g, and h) Luxol fast blue stain; b, d, and f, immunohistochemistry for TMEV antigen. Magnifications: a to f, ×75; g and h, ×150; inset, ×320.

FIG. 5

Detection of TMEV antigen-positive cells in the CNS by immunohistochemistry. In the brain (a), we detected more viral antigens in all groups during the acute phase, 1 week postinfection (p.i.) pDA virus-infected mice contained significantly more antigen-positive cells than DApB (∗∗, P < 0.01) or DApBL2M (∗, P < 0.05) virus-infected mice. During the chronic phase, while a few viral antigen-positive cells were detected in the white matter in the brainstem after pDA and DApB virus infection, viral antigen was detected in the gray matter, particularly in the pyramidal cell layer of the hippocampus after DApBL2M virus infection. In the spinal cord (b), more viral antigen-positive cells were detected during the chronic phase, 1 and 4 months after infection, in the mice infected with pDA or DApB virus. Few viral antigen-positive cells were seen in DApBL2M virus-infected mice (∗, P < 0.05 compared with pDA, analysis of variance). Values are mean antigen-positive cells per mouse ± standard error of the mean for five to six mice.

FIG. 6

Neuropathology during the chronic phase of TMEV infection. Mice were killed 4 months after pDA (a, b, and g), DApB (c and d), or DApBL2M (e, f and h to j) virus infection. pDA virus-infected mice (a) showed severe inflammation (large arrows) with demyelination in the spinal cord white matter (arrowheads) versus DApB virus-infected mice (c). pDA virus-infected mice (b) have more TMEV antigen-positive cells (small arrows) than DApB virus-infected mice (d). In DApBL2M virus-infected mice, spinal cords appeared normal (e) and no viral antigen-positive cells were detected (f). While no lesions were found in the gray matter of the brain in pDA (g) or DApB virus-infected mice, we could see hippocampus inflammation (h, large arrows) in DApBL2M virus-infected mice similar to that of acute disease, even after 4 months postinfection. During the chronic phase of DApBL2M virus infection, both viral antigen (i, diaminobenzidine stain, brown, arrow) and genome (j, BCIP-NBT, blue, arrowhead) could be detected in the pyramidal cell layer of the hippocampus, using immunohistochemistry and in situ hybridization, respectively. (a, c, e, g, and h) Luxol fast blue stain; (b, d, f, and i) immunohistochemistry for TMEV antigen; (j) in situ hybridization for TMEV genome. Magnifications: a to f, ×70; g to j, ×150.

FIG. 7

Serum anti-TMEV antibody titer from the mice infected with pDA (blank columns), DApB (hatched columns), or DApBL2M (closed columns) virus. Using ELISA, we detected serum anti-TMEV antibodies from the mice 1 week and 1 and 4 months after virus infection. Significant anti-virus antibody responses were detected in all groups as early as 1 week postinfection. During the chronic phase, while DApBL2M virus-infected mice produced lower antibody responses (∗, P < 0.05; ∗∗, P < 0.01 compared with pDA) than pDA virus-infected mice, we could detect high anti-virus antibody responses in all groups. Each experimental group consisted of six to eight mice.