Howard L. Lipton

Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler's murine encephalomyelitis virus (TMEV)

Knowledge of the cells in which Theilers murine encephalomyelitis virus (TMEV) persists is crucial to understanding the pathogenesis of TMEV-induced demyelinating disease; however, it is still uncertain whether oligodendrocytes or macrophages are the primary target for persistence. In this study, mononuclear cells (MNC) isolated directly from central nervous system (CNS) inflammatory infiltrates of TMEV-infected mice on discontinuous Percoll gradients were found to contain infectious TMEV. Macrophages appeared to be the principal MNC infected as determined by two-color immunofluorescence. Infectious center assay and double immunostaining together indicated the presence and possible synthesis of TMEV in approximately 1 in 225 to 1 in 1000 CNS macrophages, with 1 to 7 PFU produced per macrophage. On the basis of these findings, limited replication in macrophages is consistent with the total CNS virus content detected at any time during the persistent phase of the infection as well as the slow pace of the infection.

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. III. Failure of neuroantigen-specific immune tolerance to affect the clinical course of demyelination

Abstract Intracerebral inoculation of Theilers murine encephalomyelitis virus (TMEV) into susceptible mouse strains produces a chronic demyelinating disease in which mononuclear cell-rich infiltrates in the central nervous system (CNS) are prominent. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility complex (MHC) class II-restricted delayed-type hypersensitivity (DTH) responses in which lymphokine-activated macrophages mediate bystander demyelination. The present study examined the possibility that concomitant or later-appearing neuroantigen-specific autoimmune T cell responses, such as those demonstrated in chronic-relapsing experimental allergic encephalomyelitis (R-EAE), may contribute to the demyelinating process following TMEV infection. T cell responses against intact, purified major myelin proteins (myelin basic protein (MBP) and proteolipid protein (PLP), and against altered myelin constituents were readily demonstrable in SJL/J mice with R-EAE, but were not detectable in SJL/J mice with TMEV-induced demyelinating disease. TMEV-infected mice also did not display T cell responses against the peptide fragments of MBP(91–104) and PLP(139–151) recently shown to be encephalitogenic in SJL/J mice. In addition, induction of neuroantigen-specific tolerance to a heterogeneous mixture of CNS antigens, via the i.v. injection of syngeneic SJL/J splenocytes covalently coupled with mouse spinal cord homogenate, resulted in significant suppression of clinical and histologic signs of R-EAE and the accompanying MBP- and PLP-specific DTH responses. In contrast, neuroantigen-specific tolerance failed to alter the development of clinical and histologic signs of TMEV-induced demyelinating disease or the accompanying virus-specific DTH and humoral immune responses. These findings demonstrate that TMEV-induced demyelinating disease can occur in the apparent absence of neuroantigen-specific autoimmune responses. The relationship of the present results to the immunopathology of multiple sclerosis is discussed.

Chronic neurologic disease in Theiler's virus infection of SJL/J mice ☆

This study demonstrates that most SJL/J mice inoculated intracerebrally (IC) with 1000 suckling mouse 50% mean lethal doses of Theilers encephalomyelitis virus (TMEV) develop flaccid paralysis 10-21 days after infection when there is acute spinal cord gray matter involvement (early disease). Surviving mice later develop a distinctive chronic neurologic disorder which is associated with marked mononuclear cell infiltrates and active demyelination in spinal cord white matter (late disease). Moreover, about one-fourth of infected animals only develop signs of late disease which may begin after an incubation period as long as 2 and a half months. Affected mice are less active, incontinent, and have a waddling, spastic gait. Minimal stimulation induces prolonged extensor spasms of all limbs. These late-developing manifestations of chronic TMEV infection are progressive and clinical remissions have not been observed. The effect of persistent CNS infection on general development was monitored by weekly measurement of body weight; however, the growth of chronically-infected mice was found to parallel that of control animals.

Theiler’s virus antigen detected in mouse spinal cord 2½ years after infection

Spinal cord sections from mice injected with Theilers murine encephalomyelitis virus (TMEV) and surviving for 1 year and longer after infection were stained for virus antigen by two immunohistochemical techniques. Virus antigen was detected from 1 to 2½ years after infection, a time when no virus was recovered at an assay sensitivity of 50 plaqueforming units per gram of tissue. The implication this has regarding the detection of a virus in MS is discussed.

Phylogenetic Analysis of the Species Theilovirus : Emerging Murine and Human Pathogens

ABSTRACT The Cardiovirus genus of the family Picornaviridae includes two distinct species, Encephalomyocarditis virus and Theilovirus. We now report the complete nucleotide sequences of three Theilers murine encephalomyelitis virus (TMEV) strains (TO Yale, TOB15, and Vie 415HTR) and of Vilyuisk human encephalomyelitis virus (VHEV). This information, together with the recently reported sequences of divergent theiloviruses (Theilers-like rat virus [TRV] and Saffold viruses 1 and 2 [SAFV-1 and SAFV-2]), enables an updated phylogenetic analysis as well as a reexamination of several gene products important in the pathogenesis of this emerging group of viruses. In the light of the known neurotropism of TMEV and the new human SAFV-1 and SAFV-2, the resulting data suggest the existence of theiloviruses that cause human central nervous system infections. Our phylogenetic analyses point to the classification of presently known theiloviruses into five types: TMEV, VHEV, TRV, SAFV-1, and SAFV-2.

High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus

ABSTRACT The low-neurovirulence Theilers murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 × 109) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.

Apoptotic cells, including macrophages, are prominent in Theiler's virus-induced inflammatory, demyelinating lesions.

ABSTRACT Theilers 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 ∼17% remained unidentified. In situ hybridization revealed viral RNA in ∼1% of apoptotic cells.

Theiler's Murine Encephalomyelitis Virus Induces Apoptosis in Gamma Interferon-Activated M1 Differentiated Myelomonocytic Cells through a Mechanism Involving Tumor Necrosis Factor Alpha (TNF-α) and TNF-α-Related Apoptosis-Inducing Ligand

ABSTRACT Infection of susceptible mice with the low-neurovirulence Theilers murine encephalomyelitis virus strain BeAn results in an inflammatory demyelinating disease similar to multiple sclerosis. While the majority of virus antigen is detected in central nervous system macrophages (Mφs), few infiltrating Mφs are infected. We used the myelomonocytic precursor M1 cell line to study BeAn virus-Mφ interactions in vitro to elucidate mechanisms for restricted virus expression. We have shown that restricted BeAn infection of M1 cells differentiated in vitro (M1-D) results in apoptosis. In this study, BeAn infection of gamma interferon (IFN-γ)-activated M1-D cells also resulted in apoptosis but with no evidence of virus replication or protein expression. RNase protection assays of M1-D cellular RNA revealed up-regulation of Fas and the p55 chain of the tumor necrosis factor alpha (TNF-α) receptor transcripts with IFN-γ activation. BeAn infection of activated cells resulted in increased caspase 8 mRNA transcripts and the appearance of TNF-α-related apoptosis-inducing ligand (TRAIL) 4 h postinfection. Both unactivated and activated M1-D cells expressed TRAIL receptors (R1 and R2), but only activated cells were killed by soluble TRAIL. Activated cells were also susceptible to soluble FasL- and TNF-α-induced apoptosis. The data suggest that IFN-γ-activated M1-D cell death receptors become susceptible to their ligands and that the cells respond to BeAn virus infection by producing the ligands TNF-α and TRAIL to kill the susceptible cells. Unactivated cells are not susceptible to FasL or TRAIL and require virus replication to initiate apoptosis. Therefore, two mechanisms of apoptosis induction can be triggered by BeAn infection: an intrinsic pathway requiring virus replication and an extrinsic pathway signaling through the death receptors.

Enhanced detection of Theiler's virus RNA copy equivalents in the mouse central nervous system by real-time RT-PCR.

Infection of mice by low-neurovirulence Theilers murine encephalomyelitis virus (TMEV), such as BeAn and DA viruses, provides a relevant experimental animal model for multiple sclerosis (MS). As a step toward determining the kinetics of a persistent central nervous system (CNS) infection that leads to chronic demyelination, we adapted a rapid, accurate and highly specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantitation of BeAn virus RNA copy equivalents in mouse tissues. The assay enabled detection of as few as 20-30 copies of BeAn virus RNA per microg of total RNA from infected mouse tissues and results for spinal cord revealed the same high levels of BeAn RNA as detected by Northern hybridization during the first 4 months of the persistent infection, but also was able to detect virus RNA copies as late as 1 year post-infection. Real-time RT-PCR analysis of BeAn virus RNA copy equivalents in different parts of the CNS, analyses not possible by Northern hybridization, revealed the following cline of virus persistence: spinal cord>brainstem/cerebellum>cerebrospinal fluid (CSF)>cerebral hemispheres. Systemic organs, including heart, intestine and mesenteric lymph nodes of infected mice, showed no evidence of viral persistence at 4 months post-infection.

The Theiler's murine encephalomyelitis virus (TMEV) model for multiple sclerosis shows a strong influence of the murine equivalents of HLA-A, B, and C ☆

Following intracerebral inoculation of Theilers murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized histologically by mononuclear cell-rich infiltrates in the central nervous system (CNS). An immune-mediated basis for this disease is strongly supported by previous studies demonstrating a correlation between clinical disease susceptibility, the presence of particular H-2 region genotypes, and the development of chronically elevated levels of TMEV-specific, MHC class II-restricted delayed-type hypersensitivity (DTH). The present study compared disease susceptibility in (B10.S X SJL)F1 and (B10.S(26R) X SJL)F1 mice which differ only at the D region of the H-2 complex. The data conclusively demonstrates a major influence for homozygosity of H-2s alleles at the H-2D region (the murine equivalent of the human class I HLA-A, B, and C genes) in determining disease susceptibility, as measured by either clinical or histopathological endpoints. In addition, disease susceptibility strongly correlated with the development of high levels of TMEV-specific DTH in the susceptible (B10.S X SJL)F1 strain. However, disease susceptibility did not appear to correlate with TMEV titers in the CNS, TMEV-specific humoral (ELISA and neutralizing) immune responses, or virus-specific splenic T cell proliferative responses. These findings lend additional support to our hypothesis that CNS myelin damage is mediated by a TMEV-specific DTH response. The possible role of class I-restricted responses in the demyelinating process is discussed and murine TMEV-induced demyelinating disease is compared with experimental allergic encephalomyelitis as relevant animal models for human multiple sclerosis.