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Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(-/-) mice were fully permissive.

Following intracerebral inoculation, the DA strain of Theiler's virus sequentially infects neurons in the gray matter and glial cells in the white matter of the spinal cord. It persists in the latter throughout the life of the animal. Several observations suggest that the virus spreads from the gray to the white matter by axonal transport. In contrast, the neurovirulent GDVII strain causes a fatal encephalitis with lytic infection of neurons. It does not infect the white matter of the spinal cord efficiently and does not persist in survivors. The inability of this virus to infect the white matter could be due to a defect in axonal transport. Using footpad inoculations, we showed that the GDVII strain is, in fact, transported in axons. Transport was prevented by sectioning the sciatic nerve. The kinetics of transport and experiments using colchicine suggested that the virus uses microtubule-associated fast axonal transport. Our results show that a cardiovirus can spread by fast axonal transport and suggest that the inability of the GDVII strain to infect the white matter is not due to a defect in axonal transport.

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.

The strains of Theiler's murine encephalomyelitis virus, a picornavirus, are divided into two groups according to their neurovirulence after intracerebral inoculation. The highly virulent GDVII strain causes an acute, fatal encephalomyelitis, whereas the DA strain causes a mild encephalomyelitis followed by a chronic inflammatory demyelinating disease associated with viral persistence. Studies with recombinant viruses showed that the capsid plays the major role in determining these phenotypes. However, the molecular basis for the effect of the capsid on neurovirulence is still unknown. In this paper, we describe a large difference in the patterns of infection of primary neuron cultures by the GDVII and DA strains. Close to 90% of the neurons were infected 12 h after inoculation with the GDVII strain, and the cytopathic effect was complete 24 h postinoculation. In contrast, with the DA strain, viral antigens were not detected in neurons until 24 h postinoculation. Infected neurons accounted for only 2% of the total number of neurons, even 6 days after inoculation. No cytopathic effect was visible, and the cultures could be kept for the same length of time as the noninfected controls. Because the neurovirulence of the GDVII strain has been mapped to the capsid, we examined the role of the capsid in this difference of phenotype. We showed, using recombinant viruses, that the capsid was indeed responsible for the pattern of infection observed in vitro, most likely through its role in viral entry. Thus, the levels of neurovirulence of the GDVII and DA strains correlate with their abilities to infect cultured neurons, and this ability is controlled by the capsid.

Following intracranial inoculation, Theiler's virus causes either an acute encephalitis (strain GDVII) or a chronic demyelinating disease (strain DA). The DA strain sequentially infects the grey matter of the brain, the grey matter of the spinal cord, and, finally, the white matter of the spinal cord, where it persists in glial cells and causes demyelinating lesions. Analysis of the phenotype of recombinant viruses has shown that the viral capsid contains determinants for persistence and demyelination. Our previous studies showed that a Lys at position 141 of the VP2 capsid protein (VP2-141) could render a chimeric virus persistent. We also reported that another recombinant virus, virus R5, migrated from the grey matter of the brain to that of the spinal cord inefficiently and was unable to infect the white matter of the spinal cord. In this article, we report that introducing a Lys at position VP2-141 in virus R5 increases its ability to infect the white matter of the spinal cord. Our results indicate that this amino acid is important for the spread of the virus within the central nervous system.