Following intracranial inoculation, neurovirulent mouse hepatitis virus (MHV) strains induce acute inflammation, demyelination and axonal loss in the CNS. Prior studies using recombinant MHV strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. A demyelinating MHV strain induces optic neuritis, but whether this is due to retrograde axonal transport of viral particles to the retina, or if it is due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. Using recombinant isogenic MHV strains, we examined the ability of recombinant MHV to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye following intracranial inoculation. Recombinant demyelinating MHV induced macrophage infiltration of optic nerves, demyelination and axonal loss whereas optic neuritis and axonal injury were minimal in mice infected with the non-demyelinating MHV strain that differs in the spike gene. Thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. These data indicate that MHV spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for CNS viral infections and associated diseases.

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.

Demyelination in the central nervous system induced by neurovirulent strains of Mouse Hepatitis Virus (MHV) is mediated by the viral spike glycoprotein, but it is not clear whether the mechanism of this disease pathology involves direct viral infection of oligodendrocytes. Detailed studies of glial cell tropism of MHV are presented, demonstrating that direct MHV infection of oligodendrocytes differs between demyelinating (RSA59) and non-demyelinating (RSMHV2) viral strains both in vitro and in vivo. Our results indicate that direct injury of mature oligodendrocytes is an important mechanism of virus-induced demyelination. In vivo, RSA59 infection was identified in spinal cord gray and white matter, but infected oligodendrocytes were restricted to white matter. In contrast, RSMHV2 infection was restricted to gray matter neurons and was not localized to oligodendrocytes. In vitro, RSA59 can infect both oligodendrocyte precursors and differentiated oligodendrocytes, whereas RSMHV2 can infect oligodendrocyte precursors but not differentiated oligodendrocytes. Viral spreading through axonal means to white matter and release of the demyelinating strain MHV at the nerve end is critical for oligodendrocytes infection and subsequent demyelination. Understanding the mechanisms by which known viruses effect demyelination in this animal model has important therapeutic implications in the treatment of human demyelinating disease.

The isogenic host attachment spike protein recombinant demyelinating strain of mouse hepatitis virus (MHV) (RSA59) and the nondemyelinating strain (RSMHV2) differ in their abilities to infect distinct types of neural cells, spread from cell to cell, and induce subsequent demyelination and axonal loss. The differential demyelination properties of RSA59 and RSMHV2 may be a function of spike protein-mediated neuronal transport. Disruption of microtubules with colchicine and vinblastine significantly blocks neuronal transport and reduces the replication of RSA59, whereas RSMHV2 remains unaffected. R SA59 (demyelinating [DM]) and RSMHV2 (nondemyelinating [NDM]), enhanced green fluorescent protein (EGFP)-expressing recombinant strains of mouse hepatitis virus (MHV) are isogenic except for the spike gene, which encodes a glycoprotein expressed on the virion envelope that mediates many biological properties of MHV (1-5). Histopathological studies demonstrated that brain pathology from infection with either strain consists of encephalitis, characterized by parenchymal lymphocytic infiltrates and microglial activation with associated lymphocytic meningitis, but the strains differ in their abilities to induce demyelination and axonal loss. Evaluation of axonal loss and demyelination in spinal cord, where there is a clear separation of gray matter and large white-matter tracts, showed that RSA59 infection begins in the neuronal cell body, propagates centripetally to the axon, and subsequently induces axonal degeneration and demyelination (1, 3). RSMHV2 infection is mainly restricted to gray matter, is unable to spread from gray to white matter, and as a result cannot induce demyelination. In optic nerve (another white-matter tract), RSA59 induces inflammation and subsequent demyelination and axonal loss following intracranial inoculation, whereas RSMHV2 does not (3,4,6). RSMHV2 shows impaired spread in spinal cord and optic nerve compared to RSA59, highlighting the important role of spike-mediated transneuronal spread in axonal damage and demyelination. Differential spreading in the CNS could be due to interactions between the spike protein and host neuronal cytoskeleton proteins involved in anterograde and retrograde movement along the microtubule tracks, as seen in some human neurotropic viruses (7-12). Thus, the failure of NDM strains to trigger myelin damage could be entirely a function of failure of microtubule-mediated transport to the white matter. Indeed, evidence shows that the JHM strain of MHV spreads transneuronally, with MHV protein trafficking dependent on the presence of microtubules (13). Therefore, possible involvement of microtubule-mediated axonal transport of RSA59 in comparison to RSMHV2 was studied in colchicine-or vinblastine-treated Neuro2a cells to determine whether spike protein mediates microtubule-mediated neuronal transport. Colchicine and vinblastine are known microtubule-depolymerizing agents that have been shown to be effective blockers of the axonal transport system both in vivo and in vitro...

The presence of immunoglobulin oligoclonal bands in the cerebrospinal fluid of Multiple Sclerosis (MS) patients supports the hypothesis of an infectious etiology, although the antigenic targets remain elusive. Neurotropic mouse hepatitis virus (MHV) infection in mice provides a useful tool for studying mechanisms of demyelination in a virus-induced experimental model of MS. This study uses Affymetrix microarray analysis to compare differential spinal cord mRNA levels between mice infected with demyelinating and non-demyelinating strains of MHV to identify host immune genes expressed in this demyelinating disease model. The study reveals that during the acute stage of infection, both strains induce inflammatory innate immune response genes, whereas upregulation of several immunoglobulin genes during chronic stage infection is unique to infection with the demyelinating strain. Results suggest that the demyelinating strain induced an innate-immune response during acute infection that may promote switching of Ig isotype genes during chronic infection, potentially playing a role in antibody-mediated progressive demyelination even after viral clearance.

Single-walled carbon nanotubes (SWNTs) have been increasingly used as scaffolds for neuronal growth and differentiation. In this report, we have investigated how biocompatible functionalized SWNTs can affect the neuronal growth and morphology of an established neuronal cell line neuro2a (N2a). Interestingly, SWNTs covalently attached with biocompatible poly-D-lysine (PDL), facilitate neuronal growth and differentiation, neuronal growth on SWNTs with a combination of poly-ethylene glycol (PEG) with laminin (LA), a positive control molecule, is also studied. As expected, the mean length of the dendritic processes and axons is significantly larger with PDL as well as PEG-LA functionalized SWNTs compared to the control molecules. Most importantly, N2a cells retain neuronal morphology in the culture for a longer time (21 days) in presence of SWNT-PDL or SWNT-PEG LA, compared to the control culture. SWNTs modified neuronal cells in culture are infected with enhanced green fluorescent protein (EGFP) tagged recombinant neurotropic mouse hepatitis virus (MHV) to demonstrate that SWNT modulated N2a cells are a promising substrate for understanding the viral antigen spread and persistence as a proof of hypothesis.