The Role of Myelin in Theiler's Virus Persistence in the Central Nervous System
The Authors tried to find a link between the presence of the myelin in the persistence in the central nervous system by the Theiler’s murine encephalomyelitis virus (TMEV). They did this by comparing the wild-type mouse with the shiverer mouse. The shiverer mouse is a mutant with a deletion in the myelin basic protein gene (Mbp), they found previously that this mouse is resistant to persistent infection by TMEV. So they used this mouse to examine the importance of myelin in the persistence of the disease.
They knew from previous experiments that the shiverer mouse where susceptible to the “early disease” which lasts approximately two weeks after infection. The virus causes an acute encephalomyelitis with infection of neurons and to a lesser extent, of macrophages and astrocytes in the grey matter. From this point the virus is either cleared by the immune system or it persists, this persistence of the infection causes gait disorders and incontinence, and it is referred to as the “late disease”. They found that these shiverer mice were immune to this late disease. For this mutant, the resistance to persistent infection cannot be overcome by increasing viral dose. The extreme resistance of the shiverer mice indicated that the Mbp mutations interacted with essential steps in the pathway leading to viral persistence. Their experiments were designed to identify the step that leads to the persistence of a picornavirus in CNS white matter by focusing on the shiverer mutant.
Their first hypothesis stated that the resistance of the shiverer mice is not mediated by radiosensitive bone marrow-derived cells. They constructed immunological chimeras between the wild type and shiverer mice. The mice were lethally irradiated and their immune systems were reconstituted with autologous or heterologous bone marrow cells. Bone marrow grafted as well as control wild type and shiverer mice were inoculated intracerebrally with 106 plaque forming units of TMEV. The viral loads in the spinal cord were measured forty-five days postinoculation. The control mice showed the expected susceptibility and resistance of wild-type and shiverer mice. The shiverer mice that received wild type bone marrow cells remained resistant, and the wild-type mice that received mutant bone marrow remained susceptible. Therefore, the radiosensitive immune cells of the shiverer mice are not responsible for resistance to persistent infection. They also tested to make sure that since microglial cells, that secrete cytokines and chemokines which play an important role in the recruitment of inflammatory cells to the site of infection, where not responsible for the resistance to persistence. The microglial cells turn over very slowly, if at all, so they would not have been exchanged by hone marrow grafting. Wild-type and Shiverer mice were injected intracranially with 10ug of polyinosinic:polycytidilic acid. Inflammatory cells were extracted from the CNS after eighteen hours and analyzed by flow cytometry. The results show that the percentage of activated cells of monocytic origin was the same for both types of mice. They also compared the inflammatory cells present in the brain of wild-type and shiverer mice 5 days postinoculation with the virus. The results of flow cytometry show that there is the same frequency of each cell type in both types of mice. Therefore the shiverer mutant does not affect the activation of microglial cells, the recruitment of inflammatory cells and generally the adaptive bone marrow mediated immune response.
Their second hypothesis was that the early disease is not altered by the shiverer mutation. Their next experiment tested the possibility that the mutation impaired an important step of the viral life cycle in the CNS. The virus was present mainly in cortex, hippocampus and hypothalamus The pattern was the same for the wild-type and mutant mice. The macrophages were also tested between the two mice but no difference was found.
Their next hypothesis was that shiverer mutation does not alter the permissiveness of oligodendrocytes to TMEV. MBP is a major myelin protein, and it is expressed by the oligodendrocytes. These cells are infected in the late disease, it was considered that the mutation could alter their permissiveness to the virus. They used color immunoflueorescence to characterize the infection. Their results show that a single cycle of viral infection was achieved in half the oligodendrocytes. The differences between the wild type and mutant mice were not statistically significant.
Their fourth hypothesis was that the Myelin contains viral antigens during persistent infections. They looked at longitudinal and transverse frozen sections of the spinal cord of persistently infected mice. They found that there was capsid antigens forming linear patterns with the same longitudinal orientation of the axons. This confirmed that myelin sheaths may contain viral capsid antigens during persistent infection. They show that the linear pattern of viral RNA and antigens observed during persistent infection of white matter is due to the infection of myelin, more than to the presence of viral particles in the axons.
Their next idea was that the virus transported in the axons is the source of infection of myelin and oligodendrocytes. Since the virus infects neurons during early disease and is transported axonally, myelin might be exposed to axonally transported virus, and infection could spread secondarily to the cell body of the oligodendrocyte. Or, oligodendrocyte cell bodies could be infected first, and the infection could spread outwardly to the myelin sheaths. To distinguish between these two ideas they used the retina and the optic nerve which are important parts of the CNS. The ganglion cells of the retina which are adjacent to the vitreous chamber, send their axons caudally through the optic nerve where they are myelinated by oligodendrocytes. They thought that if ganglion cells could be infected and if the virus were transported in optic nerve axons, it should be possible to follow the spread of the virus from axons to glial cells. By infecting one eye only, the contralateral optic nerve could serve as a control for the axonal versus hematogenous source of the virus. Wild type mice were inoculated, sacrificed everyday and the eyes, optic nerves and brain were prepared for immunohistological examination. Cells in the intermediate layer were the first to be infected. Later, the virus spread to the layer of the ganglion cells. Viral antigens also appeared in the lateral geniculate nucleus and nowhere else in the thalamus which demonstrates axonal transport. Therefore, the source of infection of myelin and oligodendrocytes and astrocytes in the optic nerve is the virus which is transported in axons.
Their last hypothesis was the myelin is the portal of entry of the virus into the white matter oligodendrocytes. They used the shiverer mutant to distinguish between the two possibilities. That wither the virus traffics from the axon to myelin cytoplasmic channels and from there to the oligodendrocyte cell body. Or that the oligodendrocyte cell bodies could be infected by the virus diffusing from degenerating axons and the infection could spread outwardly from the cell body to the myelin. The only difference between the wild type and the mutant is that the amount of myelin is reduced. First they compared the level of virus replication in the retina of the wild type and shiverer mice. The minus strand viral RNA was found only in infected cells, so therefore they could say the assay was not biased by viral particles in the inoculum. Then they compared the time of arrival of the infection in the lateral geniculate nucleus for wild type and mutant mice using immunoflueorescence and found no difference. It can be concluded that the amount of virus transported in the optic nerve must be very similar in wild-type and shiverer mice.
They then scanned frozen sections of the optic nerves and found that 50-100% of infected cells were oligodendrocytes in wild type mice were in contrast no infected oligodendrocytes were found in the shiverer mutation.
Therefore the mutant considerably hindered the infection of oligodendrocytes by axonally transported virions.
The authors found in summary that the myelin is infected by axonally transported virus and that the infections spreads secondarily from the myelin to the oligodendrocyte cell body. This traffic is interrupted by the shiverer mutation. It can thus be deduced that infecting myelin cytoplasm is essential for the persistence of Theiler’s virus in the CNS.
Critique
I found this paper to be very interesting, their experiments were well documented and their results can be important for the medical community, because they warrant looking for a similar phenomenon in other persistent infections of the nervous system, including those that infect humans. While I enjoyed and understood the paper, it was very technical. If a reader did not have any knowledge of immunological techniques they would not understand what the authors did. They did not explain much of the principles behind the techniques in their paper. Most of their diagrams were also very technical and if the reader had no experience in these techniques it would be hard to gather any information from them. Though overall, the paper was very well done and generally easy to understand.
