Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system principally in macrophages, and infected macrophages in culture undergo apoptosis. We have detected abundant apoptotic cells in perivascular cuffs and inflammatory, demyelinating lesions of SJL mice chronically infected with TMEV. T cells comprised 74% of apoptotic cells, while 8% were macrophages, 0.6% were astrocytes, and ϳ17% remained unidentified. In situ hybridization revealed viral RNA in ϳ1% of apoptotic cells.Theiler's murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus in the family Picornaviridae, produces a central nervous system (CNS) infection in mice leading to a chronic, inflammatory demyelinating pathology. TMEV preferentially replicates in CNS macrophages-microglia (hereafter, macrophages) during persistence and to a lesser extent in oligodendrocytes and astrocytes (11,21,26). A central role of macrophages in TMEV persistence was demonstrated by depletion of peripheral macrophages with mannosylated liposomes (26). One model of persistence involves macrophageto-macrophage spread limited by a block(s) in virus replication as well as host antiviral immune responses but with dissemination to other cells, e.g., astrocytes and oligodendrocytes, where infection is cytolytic.TMEV infection of macrophages in culture is dependent on the state of macrophage differentiation and activation (14,29). Myelomonocytic precursors and activated macrophages are both resistant to infection, possibly due to the absence of coreceptors and increased innate immunity (14-16, 29; unpublished data). This limited window of susceptibility might account in part for the infection of only a small percentage of macrophages in the mouse spinal cord (11). Once infected, macrophages (both in mice and in cell culture) restrict TMEV replication at the level of viral assembly (11,14,18,23).TMEV-infected macrophages in culture undergo apoptosis (14,23,24,29), either through an intrinsic pathway in nonactivated macrophages that requires virus replication and results in caspase activation or through an extrinsic pathway in gamma interferon-activated macrophages that involves signaling through receptors for tumor necrosis factor alpha and tumor necrosis factor alpha-related apoptosis-inducing ligand (16). However, the only report of apoptosis in TMEV infection of mice indicated that terminal deoxynucleotidyltransferase (TdT)-mediated UTP nick end labeling (TUNEL)-positive cells were seldom observed during viral persistence in the CNS (34), which suggests that apoptosis may be of little importance. Here, we report the detection of numerous apoptotic cells, primarily in the white matter in inflammatory demyelinating lesions in spinal cord sections of SJL mice chronically infected with BeAn virus.Quantitation of apoptotic cells in spinal cords of mice with demyelinating disease. Cells in the spinal cords of 12 SJL mice inoculated intracerebrally with 10 6 PFU of BeAn virus and undergoing demyelination were assessed for apoptosis b...
The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10-to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.Theiler's murine encephalomyelitis virus (TMEV) is a naturally occurring enteric pathogen of mice that belongs to the Cardiovirus genus in the Picornaviridae family (39, 41). Lowneurovirulence strains, such as BeAn and DA, produce persistent infections in the central nervous systems (CNS) of susceptible strains of mice that result in mononuclear cell inflammation and largely immune system-mediated demyelination, providing an experimental analog for multiple sclerosis (MS) in humans (6,13,19,44). While only small amounts of infectious virus (100 to 1,000 PFU/spinal cord) are recovered during the persistent phase of infection (11, 29), very high levels of viral genome equivalents have been reported (49), suggesting that widespread and persistent TMEV infection involves continuous viral replication with restricted infectious virus production. Viral antigens and RNA are largely found in neurons in the brain and spinal cord during the acute phase of infection (2, 16), whereas macrophages or microglia containing viral antigen(s) and RNA predominate in the spinal cord during chronic infection (3, 30). The spread of infection to oligodendrocytes and astrocytes is also seen (3,9,44,47,51). TMEV replication in macrophages is highly restricted at the levels of RNA replication and virion assembly, consistent with mechanisms of persistence of cytolytic RNA viruses (23, 49). The mechanism(s) underlying TMEV transition from an acute neuronal infection in the gray matter to a chronic macrophagic and glial infection in the white matter is not well u...
The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HSdeficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.Theiler's murine encephalomyelitis viruses (TMEV) are divided into two groups based on neurovirulence following intracerebral (i.c.) inoculation of mice. High-neurovirulence strains, such as GDVII, produce fatal encephalitis in mice, while low-neurovirulence strains, such as BeAn and DA, induce early poliomyelitis followed by persistent infection in the central nervous system (CNS) and demyelinating disease in mice (26,28). Members of both neurovirulence groups are 90% identical in nucleotide sequences, 95% identical in amino acid contents, and differ only slightly in their overall threedimensional structures (31). Despite their close genetic and structural relatedness, these viruses differ significantly in their mode of binding to mammalian cells (35,39) and in their association with intracellular membranes during replication (8).TMEV use at least two molecules for attachment and cell entry (19). Recent studies have shown that low-neurovirulence strains bind ␣2,3 sialic acid moieties on N-linked oligosaccharides (7,39,42), while high-neurovirulence strains attach to the proteoglycan heparan sulfate (HS) (35). Brain-derived stocks of GDVII virus bind HS (35), indicating that this interaction is not an artifact of cell culture adaptation of the virus. Since TMEV are picornaviruses, nonenveloped viruses which are unable to fuse with the lipid bilayer of cells, internalization is believed to require a specific protein entry receptor. Viruses of both TMEV neurovirulence groups have been shown to bind an as yet unidentified sialylated 34-kDa protein in a ligand (virus) protein overlay assay (21, 27).To gain further insight into cell entry by TMEV, we focused on the interaction between high-neurovirulence GDVII and HS by using GDVII virus adapted to proteoglycan-deficient cells. The adapted virus, designated GD-A745, acquired two mutations, both on surface loops in the capsid. GD-A745 no longer used HS for binding and entry, and its neurovirulence in mice was attenuated compared to parental GDVII. Neuronal tropism in GD-A745-infected adult mice changed from cereb...
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