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

The low-neurovirulence Theiler's 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 x 10(9)) 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.     

Gamma interferon is critical for neuronal viral clearance and protection in a susceptible mouse strain following early intracranial Theiler's murine encephalomyelitis virus infection

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-gamma(-/-) H-2(q) mice. IFN-gamma deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.     

Immunoglobulins and complement in demyelination induced in mice by Theiler's virus

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) produces chronic demyelination and persistent infection in the central nervous system (CNS) of susceptible SJL mice. This series of experiments examined the contribution of humoral immunity and C to myelin destruction. As in multiple sclerosis, mice persistently infected with TMEV had elevated levels of IgG and oligoclonal bands in the cerebrospinal fluid (CSF). Immunoblot studies revealed that even in animals exhibiting profound demyelination, IgG in the serum and CSF was directed primarily at virus antigen rather than at normal myelin components. Inflammatory cells positive for Ig were distributed mainly around blood vessels, but occasionally they infiltrated the spinal cord parenchyma. Rare examples of myelin sheaths positive for IgG were found by immunoelectron microscopy in spinal cord sections from infected mice; the third component of complement (C3) was commonly found in the walls of CNS blood vessels but not on myelin. Neither serum nor CSF IgG from infected mice bound to myelin sheaths or other CNS components in sections of normal syngeneic spinal cord. There were significantly more demyelinating lesions in infected mice depleted of C components with cobra venom factor. These data do not support a humoral autoimmune basis for the CNS demyelination that occurs in association with persistent TMEV infection. However, the humoral immune response directed at TMEV antigens may either limit virus spread or promote virus persistence.     

Induction of autoreactive CD8+ cytotoxic T cells during Theiler's murine encephalomyelitis virus infection: implications for autoimmunity

Theiler's murine encephalomyelitis virus (TMEV) belongs to the family Picornaviridae and causes demyelinating disease in the spinal cords of infected mice. Although immune responses have been shown to play an important role in demyelination, the precise effector mechanism(s) is unknown. Potentially autoreactive cytotoxic cells could contribute to the destruction. We tested whether an autoreactive cell induced by TMEV infection mediated cytotoxicity by using a 5-h (51)Cr release assay in SJL/J mice. Spleen cells from TMEV-infected mice were stimulated with irradiated TMEV antigen-presenting cells and used as effector cells. The effector cells differed from conventional cytotoxic T cells since these cells could kill both TMEV-infected and uninfected syngeneic or semisyngenic cell lines (PSJLSV and BxSF11gSV) but could not kill an allogeneic cell line (C57SV). The TMEV-induced autoreactive cells were also different from conventional natural killer (NK) cells or lymphokine-activated killer (LAK) cells, because they could kill neither NK cell-sensitive YAC-1 nor NK cell-resistant P815 and EL4 cells. Induction of autoreactive cells was not detected in vaccinia virus infection. The autoreactive killing required direct cell-to-cell contact and was mediated by a Fas-FasL pathway but not by a perforin pathway. The phenotype of the killer cells was CD3(+) CD4(-) CD8(+). Intracerebral inoculation of the effector cells into naive mice caused meningitis and perivascular cuffing not only in the brain parenchyma but also in the spinal cord, with no evidence of viral antigen-positive cells. This is the first report demonstrating that TMEV can induce autoreactive cytotoxic cells that induce central nervous system pathology.     

ts1, a Paralytogenic mutant of Moloney murine leukemia virus TB, has an enhanced ability to replicate in the central nervous system and primary nerve cell culture.

A temperature-sensitive mutant of Moloney murine leukemia virus TB (MoMuLV-TB), ts1, which is defective in intracellular processing of envelope precursor protein (Pr80env), also possesses the ability to induce hind-limb paralysis in infected mice. To investigate whether ts1 has acquired neurotropism and to determine to what extent it can replicate in the central nervous system, we compared viral titers in the spleen, plasma, spinal cord, and brain throughout the course of infection of mice infected with ts1 and parental wild-type (wt) MoMuLV-TB. In both the ts1- and wt-inoculated mice, the concentrations of infectious virus recovered from the plasma and spleen increased rapidly and reached a plateau by 10 days postinfection (p.i.). In contrast, virus concentrations in the spinal cord and brain of ts1-inoculated mice increased gradually and reached a titer comparable to that in the spleen and exceeding that in the plasma only at 25 to 30 days p.i. At this time, the virus titer was approximately 200X greater in ts1-infected spinal cord tissue and approximately 20X greater in ts1-infected brain tissue than in the same wt-infected tissues. Paralysis became evident at 25 to 30 days p.i. in ts1-inoculated mice, whereas the wt-inoculated mice were normal. In addition, a substantial amount of Pr80env was detected in the spinal cords of ts1-inoculated mice compared with that found in the spinal cords of wt-inoculated mice. The infectious virus isolated from ts1-infected nerve tissue was found to possess the characteristic phenotype of the ts1 virus. Microscopic lesions of ts1-inoculated mice at 30 days p.i. consisted of vacuolar degeneration of motor neurons and spongy change of white matter in the brain stem and spinal cord. Similar but less severe lesions were observed in wt-inoculated mice. With primary cultures of central nervous system tissue we showed that ts1 can infect and replicate in both neuron and glial cells. In contrast, although wt MoMuLV-TB replicated in glial cell-rich culture, viral replication was barely detectable in neuron-rich culture.     

Theiler's murine encephalomyelitis virus (TMEV) subgroup strain-specific infection in neural and non-neural cell lines.

GDVII subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. In contrast, DA subgroup strains are less virulent and establish a persistent central nervous system infection which results in demyelinating disease. We previously reported a subgroup-specific infection in a macrophage-like cell line, J774-1 cells; i.e., GDVII strain does not replicate in J774-1 cells, whereas the DA strain actively replicates in these cells. In addition, this subgroup-specific virus growth is shown to be related to the presence of L* protein, a 17 kDa protein translated out-of-frame of the viral polyprotein from an AUG located 13 nucleotides downstream from the polyprotein's AUG. The present paper demonstrated that this subgroup-specific infection is observed in murine monocyte/macrophage lineage cell lines, but not in other murine cell lines including neural cells. An RNase protection assay also suggested that L* protein-related virus growth is regulated at the step of viral RNA replication. As macrophages are reported to be the major cell harboring virus during the chronic demyelinating stage, the activity of L* protein with respect to virus growth in macrophages may be a key factor in clarifying the mechanism(s) of TMEV persistence, which is probably a trigger to spinal cord demyelination.     

Transgenic expression of Theiler's murine encephalomyelitis virus genes in H-2(b) mice inhibits resistance to virus-induced demyelination

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.     

Differences between C57BL/6 and BALB/cBy mice in mortality and virus replication after intranasal infection with neuroadapted Sindbis virus

Neuroadapted Sindbis virus (NSV), given intranasally, caused fatal encephalitis in 100% of adult C57BL/6 mice and 0% of BALB/cBy mice. Most C57BL/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while BALB/cBy mice did not. In situ hybridization for detecting NSV RNA and immunohistochemistry for detecting NSV antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. Virus antigen was more abundant and infectious virus increased more rapidly and reached higher levels in C57BL/6 mice than in BALB/cBy mice. Surprisingly, infectious virus was cleared faster in C57BL/6 mice, and this was associated with more rapid production of neutralizing antibody. However, viral RNA was cleared more slowly in C57BL/6 mice. In both mouse strains, more infectious virus was present in the lumbar spinal cord than in the cervical spinal cord. These data suggest that genetic susceptibility to fatal NSV encephalomyelitis is determined at least in part by the efficiency of viral replication and spread in the central nervous system. The differences identified in this study provide possible phenotypes for mapping genetic loci involved in susceptibility.     

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.     

Critical role for protein tyrosine phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler's murine encephalomyelitis virus

We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.     

A neutralization-resistant Theiler's virus variant produces an altered disease pattern in the mouse central nervous system.

Theiler's murine encephalomyelitis virus infection of mice is an animal model for human demyelinating diseases. To further define the role of this virus in the disease process, we selected a virus variant resistant to neutralization by a monoclonal antibody to VP-1. This virus variant was then injected into SJL/J mice. Central nervous system tissue was compared between variant virus- and wild-type virus-infected mice. Within the brain, no large differences were observed between the two groups as to the distribution of inflammatory infiltrates around the injection site and the number of viral antigen-positive cells during the first weeks of the observation period. In contrast, in the spinal cord major differences were found between variant virus- and wild-type virus-infected mice regarding the number of inflammatory lesions, infected cells, and the size of the areas involved with time. By immunohistochemistry, equivalent numbers of infected cells could be found in the spinal cord 1 week postinfection (p.i.): however, after that time, the number of infected cells in the wild-type virus-infected mice continued to increase, whereas the virus-positive cells from the variant virus-infected mice gradually decreased. Thus, the number of viral antigen-containing cells peaked by 1 week p.i. in the variant virus-infected animals. Conversely, the number of infected cells in the spinal cords from mice inoculated with wild-type virus steadily increased until 8 weeks p.i. At this time (8 weeks p.i.), no more variant virus antigen-positive cells could be observed within the spinal cord. Plaque assay of central nervous system tissue confirmed these differences between the two groups observed by immunohistochemistry. No infectious variant virus could be isolated after 2 weeks p.i. from the brain and 4 weeks p.i. from the spinal cord, whereas infectious wild-type virus could be detected up to the end of the observation period (12 weeks p.i.). Virus which was isolated from variant virus-infected mice still retained the neutralization-resistant phenotype. These studies emphasize the important biological in vivo activity of Theiler's virus VP-1 in determining neurovirulence.     

Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system

Theiler's murine encephalomyelitis viruses (TMEV) are ubiquitous pathogens of mice, producing either rapidly fatal encephalitis (high-neurovirulence strains) or persistent central nervous system infection and inflammatory demyelination (low-neurovirulence strains). Although a protein entry receptor has not yet been identified, carbohydrate co-receptors that effect docking and concentration of the virus on the cell surface are known for both TMEV neurovirulence groups. Low-neurovirulence TMEV use α2,3-linked N-acetylneuramic acid (sialic acid) on an N-linked glycoprotein, whereas high-neurovirulence TMEV use the proteoglycan heparan sulfate (HS) as a co-receptor. While the binding of low-neurovirulence TMEV to sialic acid can be inhibited completely, only a third of the binding of high-neurovirulence TMEV to HS is inhibitable, suggesting that high-neurovirulence strains use another co-receptor or bind directly to the putative protein entry receptor. Four amino acids on the surface (VP2 puff B) of low-neurovirulence strains make contact with sialic acid through non-covalent hydrogen bonds. Since these virus residues are conserved in all TMEV strains, the capsid conformation of this region is probably responsible for sialic acid binding. A persistence determinant that maps within the virus coat using recombinant TMEV is also conformational in nature. Low-neurovirulence virus variants that do not bind to sialic acid fail to persist in the central nervous system of mice, indicating a role for sialic acid binding in TMEV persistence. Analysis of high-neurovirulence variants that do not bind HS demonstrates that HS co-receptor usage influences neuronal tropism in brain, whereas, the HS co-receptor use is not required for the infection of spinal cord anterior horn cells associated with poliomyelitis.     

Cellular immunity in chronic Theiler's virus central nervous system infection.

After (IC) inoculation of the DA strain of TMEV, SJL/J mice develop chronic CNS infection with marked mononuclear cell infiltration of spinal cord leptomeninges and white matter and concomitant demyelination. In the present study the temporal course of cell-mediated and humoral immune responses to virus were measured in this infection. It was shown that chronic TMEV infection is associated with the development of immunologically specific spleen cell reactivity as judged by in vitro incorporation of 3H-TdR into DNA in response to inactivated TMEV antigen. Spleen cell reactivity is first detectable about 2 months after infection, persists for at least 1 year, and correlates with the temporal development of serum-neutralizing antibody. The late development of sensitized spleen cells is not the result of an immunosuppressive effect of this virus infection since infected mice exhibit normal spleen cell proliferative responses to T cell mitogens and produce normal antibody responses to a heterologous protein antigen, sheep red blood cells. In addition, anti-viral antibody inhibits virus-induced spleen cell reactivity. Finally, the antigen-reactive lymphocyte subpopulation within the spleen responsible for proliferation to TMEV antigen are T cells and not B cells.     

Differential gene expression in the EphA4 knockout spinal cord and analysis of the inflammatory response following spinal cord injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips?. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury.     

CD3+/TCR-αβ− Cells Are Important in Protecting Spinal Cord Tissues against Theiler's Virus Strain GD VII Infection

Intravenous infection with Theiler's virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN-γ produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4⁺ cells nor CD8⁺ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3⁺/TCR-γδ⁺ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN-γ as a result of stimulation by immobilized anti-CD3 mAb. Elimination of CD3⁺ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN-γ. Depletion of TCR-αβ⁺ cells and ASGM1⁺ cells did not affect the viral replication, and did not alter the production of IFN-γ. Therefore, CD3⁺/TCR-αβ^– cells producing IFN-γ play an important role in the protection of the spinal cord against Theiler's virus infection. These results suggest that CD3⁺/TCR-αβ^– cells might be identical to TCR-γδ⁺ cells.     

Central nervous system pathology caused by autoreactive CD8+ T-cell clones following virus infection

Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease in infected mice which has similarities to multiple sclerosis. Spleen cells from TMEV-infected SJL/J mice stimulated with antigen-presenting cells infected with TMEV resulted in a population of autoreactive CD8+ cytotoxic T cells that kill uninfected syngeneic cells. We established CD8+ T cell clones that could kill both TMEV-infected and uninfected syngeneic targets, although infected target cells were killed more efficiently. The CD8+ T-cell clones produced gamma interferon when incubated with either infected or uninfected syngeneic target cells. Intracerebral injection of the clones into na?ve mice induced degeneration, not only in the brain, but also in the spinal cord. This suggests that CD8+ Tc1 cells could play a pathogenic role in central nervous system inflammation.     

L* Protein of the DA Strain of Theiler’s Murine Encephalomyelitis Virus Is Important for Virus Growth in a Murine Macrophage-Like Cell Line

Strain GDVII and other members of the GDVII subgroup of Theiler’s murine encephalomyelitis virus (TMEV) are highly virulent and cause acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. On the other hand, strain DA and other members of the TO subgroup of TMEV are less virulent and establish a persistent infection in the spinal cord, which results in a demyelinating disease. We previously reported that GDVII does not actively replicate in a murine macrophage-like cell line, J774-1, whereas DA strain productively infects these cells (M. Obuchi, Y. Ohara, T. Takegami, T. Murayama, H. Takada, and H. Iizuka, J. Virol. 71:729–733, 1997). In the present study, we used recombinant viruses between these strains of the two subgroups to demonstrate that the DA L coding region of DA strain is important for virus growth in J774-1 cells. Additional experiments with a mutant virus indicate that L* protein, which is synthesized out of frame with the polyprotein from an additional alternative initiation codon in the L coding region of TO subgroup strains, is a key determinant responsible for the cell-type-specific restriction of virus growth. L* protein may play a critical role in the DA-induced restricted demyelinating infection by allowing growth in macrophages, a major site for virus persistence.