The L-coding region of the DA strain of Theiler's murine encephalomyelitis virus causes dysfunction and death of myelin-synthesizing cells

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.     

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.     

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.     

Strains from both Theiler's virus subgroups encode a determinant for demyelination.

The GDVII strain and other members of the GDVII subgroup of Theiler's murine encephalomyelitis viruses (TMEV) cause an acute lethal neuronal infection in mice, whereas the DA strain and other members of the TO subgroup of TMEV cause a chronic demyelinating disease associated with a persistent virus infection. We used GDVII/DA chimeric infectious cDNAs to produce intratypic recombinant viruses in order to clarify reasons for the TMEV subgroup-specific difference in demyelinating activity. We found that both the GDVII and DA strains contain a genetic determinant(s) for demyelinating activity. No demyelination occurs following GDVII strain inoculation because this strain produces an early neuronal disease that kills mice before white matter disease and persistent infection can occur.     

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.     

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.     

Anticapsid immunity level, not viral persistence level, correlates with the progression of Theiler's virus-induced demyelinating disease in viral P1-transgenic mice

Intracranial infection of Theiler's murine encephalomyelitis virus (TMEV) induces demyelination and a neurological disease in susceptible SJL/J (SJL) mice that resembles multiple sclerosis. While the virus is cleared from the central nervous system (CNS) of resistant C57BL/6 (B6) mice, it persists in SJL mice. To investigate the role of viral persistence and its accompanying immune responses in the development of demyelinating disease, transgenic mice expressing the P1 region of the TMEV genome (P1-Tg) were employed. Interestingly, P1-Tg mice with the B6 background showed severe reductions in both CD4(+) and CD8(+) T-cell responses to capsid epitopes, while P1-Tg mice with the SJL background displayed transient reductions following viral infection. Reduced antiviral immune responses in P1-Tg mice led to >100- to 1,000-fold increases in viral persistence at 120 days postinfection in the CNS of mice with both backgrounds. Despite the increased CNS TMEV levels in these P1-Tg mice, B6 P1-Tg mice developed neither neuropathological symptoms nor demyelinating lesions, and SJL P1-Tg mice developed significantly less severe TMEV-induced demyelinating disease. These results strongly suggest that viral persistence alone is not sufficient to induce disease and that the level of T-cell immunity to viral capsid epitopes is critical for the development of demyelinating disease in SJL mice.     

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.     

Cytotoxic T cells isolated from the central nervous systems of mice infected with Theiler''s virus.

Intracerebral inoculation of resistant mice (C57BL/10SNJ) with Theiler's murine encephalomyelitis virus (TMEV) results in acute encephalitis followed by subsequent clearance of virus from the central nervous system (CNS). In contrast, infection of susceptible mice (SJL/J) results in virus persistence and chronic immune-mediated demyelination. Both resistance and susceptibility to TMEV-induced disease appear to be immune mediated, since immunosuppression results in enhanced encephalitis in resistant mice but diminished demyelination in susceptible mice. The purpose of these experiments was to determine whether anti-TMEV cytotoxic T lymphocytes (CTLs) are generated during acute and chronic TMEV infection. Nonspecific lectin-dependent cellular cytotoxicity was used initially to detect the cytolytic potential of lymphocytes infiltrating the CNS irrespective of antigen specificity. Using TMEV-infected targets, H-2-restricted TMEV-specific CTLs of the CD8+ phenotype were demonstrated in lymphocytes from the CNS of susceptible and resistant mice, arguing against the hypothesis that the ability to generate CD8+ CTLs mediates resistance. In chronically infected SJL/J mice, TMEV-specific CTL activity was detected in the CNS as late as 226 days postinfection. These experiments demonstrate that virus-specific CTLs are present in the CNS during both acute and chronic TMEV infection. Anti-TMEV CTLs in the CNS of chronically infected SJL/J mice may play a role in demyelination through their ability to lyse TMEV-infected glial cells.     

Sialylation of the host receptor may modulate entry of demyelinating persistent Theiler's virus

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus of the Cardiovirus genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 A. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.     

Differences in avidity and epitope recognition of CD8(+) T cells infiltrating the central nervous systems of SJL/J mice infected with BeAn and DA strains of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection induces immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infectious model for human multiple sclerosis. To investigate the pathogenic mechanisms, two strains of TMEV (DA and BeAn), capable of inducing chronic demyelination in the central nervous system (CNS), have primarily been used. Here, we have compared the T-cell responses induced after infection with DA and BeAn strains in highly susceptible SJL/J mice. CD4(+) T-cell responses to known epitopes induced by these two strains were virtually identical. However, the CD8(+) T-cell response induced following DA infection in susceptible SJL/J mice was unable to recognize two of three H-2K(s)-restricted epitope regions of BeAn, due to single-amino-acid substitutions. Interestingly, T cells specific for the H-2K(s)-restricted epitope (VP1(11-20)) recognized by both strains showed a drastic increase in frequency as well as avidity after infection with DA virus. These results strongly suggest that the level and avidity of virus-specific CD8(+) T cells infiltrating the CNS could be drastically different after infection with these two strains of TMEV and may differentially influence the pathogenic and/or protective outcome.     

Regulatory role of CD1d in neurotropic virus infection

The GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) causes an acute fatal polioencephalomyelitis in mice. Infection of susceptible mice with the DA strain of TMEV results in an acute polioencephalomyelitis followed by chronic immune-mediated demyelination with virus persistence in the central nervous system (CNS); DA virus infection is used as an animal model for multiple sclerosis. CD1d-restricted natural killer T (NKT) cells can contribute to viral clearance and regulation of autoimmune responses. To investigate the role of CD1d in TMEV infection, we first infected CD1d-deficient mice (CD1^−/−) and wild-type BALB/c mice with GDVII virus. Wild-type mice were more resistant to virus than CD1^−/− mice (50% lethal dose titers: wild-type mice, 10 PFU; CD1^−/− mice, 1.6 PFU). Wild-type mice had fewer viral antigen-positive cells with greater inflammation in the CNS than CD1^−/− mice. Second, an analysis of DA virus infection in CD1^−/− mice was conducted. Although both wild-type and CD1^−/− mice had similar clinical signs during the first 2 weeks after infection, CD1^−/− mice had an increase in neurological deficits over those observed in wild-type mice at 3 to 5 weeks after infection. Although wild-type mice had no demyelination, 20 and 60% of CD1^−/− mice developed demyelination at 3 and 5 weeks after infection, respectively. TMEV-specific lymphoproliferative responses, interleukin-4 (IL-4) production, and IL-4/gamma interferon ratios were higher in CD1^−/− mice than in wild-type mice. Thus, CD1d-restricted NKT cells may play a protective role in TMEV-induced neurological disease by alteration of the cytokine profile and virus-specific immune responses.     

Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus

Microglia are resident central nervous system (CNS) macrophages. Theiler's murine encephalomyelitis virus (TMEV) infection of SJL/J mice causes persistent infection of CNS microglia, leading to the development of a chronic-progressive CD4(+) T-cell-mediated autoimmune demyelinating disease. We asked if TMEV infection of microglia activates their innate immune functions and/or activates their ability to serve as antigen-presenting cells for activation of T-cell responses to virus and endogenous myelin epitopes. The results indicate that microglia lines can be persistently infected with TMEV and that infection significantly upregulates the expression of cytokines involved in innate immunity (tumor necrosis factor alpha, interleukin-6 [IL-6], IL-18, and, most importantly, type I interferons) along with upregulation of major histocompatibility complex class II, IL-12, and various costimulatory molecules (B7-1, B7-2, CD40, and ICAM-1). Most significantly, TMEV-infected microglia were able to efficiently process and present both endogenous virus epitopes and exogenous myelin epitopes to inflammatory CD4(+) Th1 cells. Thus, TMEV infection of microglia activates these cells to initiate an innate immune response which may lead to the activation of naive and memory virus- and myelin-specific adaptive immune responses within the CNS.     

A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination

We describe here a mouse line bearing a bovine keratin K5Cre recombinase transgene. These mice showed a dual pattern of Cre-mediated recombination, depending on the parent transmitting the transgene. In paternal transmission, recombination occurred specifically in the skin and stratified epithelia-as expected according to the expression of endogenous keratin K5. However, constitutive recombination between loxP sites transmitted by the sperm took place when the mother possessed the K5Cre transgene, even when the transgene was absent in the progeny. Cre expression in late-stage oocytes, with the Cre protein persisting into the developing embryo, leads to the constitutive recombination observed. Thus, this transgenic line allows for both tissue-specific and generalized recombination, depending on the breeding scheme.     

In vitro Cre/loxP system in cells from developing gonads: investigation of the Sry promoter

There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.     

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.     

Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.     

Theiler's virus induces the MIP-2 chemokine (CXCL2) in astrocytes from genetically susceptible but not from resistant mouse strains

The murine encephalomyelitis virus of Theiler (TMEV) induces demyelination in susceptible strains of mice by a CD4(+) Th1 T cell mediated immunopathologic process. We focused on the production of one chemokine, the macrophage inflammatory protein-2 (MIP-2 or CXCL2), by cultured mouse astrocytes infected with the BeAn strain of TMEV. Analysis of a murine genome DNA hybridized with cRNA from mock- and TMEV-infected astrocytes, revealed up-regulation of three sequences encoding MIP-2. Northern blot analysis indicated increased MIP-2 mRNA expression. Levels of MIP-2 in the supernatants of infected cells as detected by ELISA, varied directly with the multiplicity of infection used. This secreted CXCL2 was biologically active inducing chemoattraction of neutrophils but not of lymphocytes. CXCL2 was specifically induced by TMEV infection, since induction was inhibited by anti TMEV antibodies. The inflammatory cytokines, IL-1alpha and TNF-alpha, which are also induced in astrocytes by TMEV, were very potent inducers of CXCL2. Nevertheless, both mechanisms of induction follows different pathways as antibodies to both cytokines fails to inhibit TMEV-induced CXCL2 up-regulation. Sera from TMEV-infected SJL/J mice with chronic demyelination, but not from BALB/c TMEV-resistant mice, revealed CXCL2 at the peak of clinical disease. Our main novel finding is the strain-dependent differences in CXCL2 expression both in vitro and in vivo. This suggest an role for this chemokine in attracting immune cells within the CNS, which in turn, might trigger demyelination in this experimental model of MS.     

CD28 costimulatory blockade exacerbates disease severity and accelerates epitope spreading in a virus-induced autoimmune disease

Theiler's murine encephalomyelitis virus (TMEV) is a natural mouse pathogen which causes a lifelong persistent infection of the central nervous system (CNS) accompanied by T-cell-mediated myelin destruction leading to chronic, progressive hind limb paralysis. TMEV-induced demyelinating disease (TMEV-IDD) is considered to be a highly relevant animal model for the human autoimmune disease multiple sclerosis (MS), which is thought to be initiated as a secondary consequence of a virus infection. Although TMEV-IDD is initiated by virus-specific CD4(+) T cells targeting CNS-persistent virus, CD4(+) T-cell responses against self myelin protein epitopes activated via epitope spreading contribute to chronic disease pathogenesis. We thus examined the ability of antibodies directed against B7 costimulatory molecules to regulate this chronic virus-induced immunopathologic process. Contrary to previous studies showing that blockade of B7-CD28 costimulatory interactions inhibit the initiation of experimental autoimmune encephalomyelitis, treatment of SJL mice at the time of TMEV infection with murine CTLA-4 immunoglobulin or a combination of anti-B7-1 and anti-B7-2 antibodies significantly enhanced clinical disease severity. Costimulatory blockade inhibited early TMEV-specific T-cell and antibody responses critical in clearing peripheral virus infection. The inhibition of virus-specific immune responses led to significantly increased CNS viral titers resulting in increased damage to myelin-producing oligodendrocytes. Following clearance of the costimulatory antagonists, epitope spreading to myelin epitopes was accelerated as a result of the increased availability of myelin epitopes leading to a more severe chronic disease course. Our results raise concern about the potential use of B7-CD28 costimulatory blockade to treat human autoimmune diseases potentially associated with acute or persistent virus infections.