Direct comparison of demyelinating disease induced by the Daniel's strain and BeAn strain of Theiler's murine encephalomyelitis virus

We compared CNS disease following intracerebral injection of SJL mice with Daniel's (DA) and BeAn 8386 (BeAn) strains of Theiler's murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.     

Mechanism of Theiler's virus-induced demyelination in nude mice

In its natural murine host, infection with Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, progressive demyelinating disease. To help elucidate the role of host immune mechanisms involved in demyelination, we studied TMEV infection in Nude mice. These animals demonstrated rising titers of infectious virus within the central nervous system and failed to produce anti-TMEV antibody. Neurologic signs including the development of severe hind limb paralysis were evident approximately 2 weeks postinfection with most animals succumbing within the first month. Immunoperoxidase studies demonstrated viral antigen in the cytoplasm of neurons and glial cells for the entire period of observation. Plaques of demyelination associated with scanty inflammatory infiltrates were present in the spinal cord by 14 days postinfection. Electron microscopic studies of the involved white matter revealed numerous degenerating glial cells, many of which contained paracrystalline arrays of picornavirus within their cytoplasm. Some of the infected glial cells were identified as oligodendrocytes by demonstrating their myelin-plasma membrane connections. The studies indicate that in Nude mice TMEV causes a lytic infection of oligodendrocytes producing demyelination independent of the T lymphocyte immune system.     

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide

Theiler's murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the D(b) gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/D(b) mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H-2(b) haplotype mount a vigorous H-2D(b)-restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contribution of the immunodominant T cell population in resistance to TMEV, FVB/D(b) mice were depleted of VP2-specific CD8(+) T cells by peptide treatment prior to virus infection. Peptide-treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the D(b)-restricted CD8(+) T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8(+) T cells.     

Genetic deletion of a single immunodominant T-cell response confers susceptibility to virus-induced demyelination

An important question in neuropathology involves determining the antigens that are targeted during demyelinating disease. Viral infection of the central nervous system (CNS) leads to T-cell responses that can be protective as well as pathogenic. In the Theiler's murine encephalomyelitis virus (TMEV) model of demyelination it is known that the immune response to the viral capsid protein 2 (VP2) is critical for disease pathogenesis. This study shows that expressing the whole viral capsid VP2 or the minimal CD8-specific peptide VP2(121-130) as "self" leads to a loss of VP2-specific immune responses. Loss of responsiveness is caused by T cell-specific tolerance, as VP2-specific antibodies are generated in response to infection. More importantly, these mice lose the CD8 T-cell response to the immunodominant peptide VP2(121-130), which is critical for the development of demyelinating disease. The transgenic mice fail to clear the infection and develop chronic demyelinating disease in the spinal cord white matter. These findings demonstrate that T-cell responses can be removed by transgenic expression and that lack of responsiveness alters viral clearance and CNS pathology. This model will be important for understanding the mechanisms involved in antigen-specific T-cell deletion and the contribution of this response to CNS pathology.     

Deletion of Beta-2-Microglobulin Ameliorates Spinal Cord Lesion Load and Promotes Recovery of Brainstem NAA Levels in a Murine Model of Multiple Sclerosis

We used genetic deletion of β2-microglobulin to study the influence of CD8(+) T cells on spinal cord demyelination, remyelination, axonal loss and brainstem N-acetyl aspartate levels during the acute and chronic phases of Theiler's murine encephalomyelitis virus (TMEV) infection. We used β2m(-/-) and β2m(+/+) B10.Q mice (of H-2(q) background) normally susceptible to TMEV-induced demyelination. Over the disease course, β2m(+/+) mice had increasing levels of demyelination and minimal late-onset remyelination. In contrast, β2m(-/-) mice had steady levels of demyelination from 45-390?dpi and remyelination was extensive and more complete. Early in the disease, brainstem NAA levels drop in both strains, but accordingly with remyelination and axonal preservation, NAA recover in β2m(-/-) mice despite equivalent brainstem pathology. At 270?dpi, β2m(+/+) mice had significantly fewer spinal cord axons than β2m(-/-) mice (up to 28% less). In addition, β2m(+/+) mice lost axons of all calibers, whereas β2m(-/-) mice had a modest loss of only medium- and large-caliber axons. This study further supports the hypothesis that CD8(+) T cells are involved in demyelination, and axonal loss following Theiler's virus-induced demyelination.     

Contrasting effects of immunosuppression on Theiler''s virus infection in mice.

In the present study, cyclophosphamide and rabbit anti-mouse thymocyte serum were used to immunosuppress SJL/J mice infected with Theiler's mouse encephalomyelitis virus (TMEV) in order to delineate the potential mechanism(s) of virus-induced cellular injury in this infection. Whereas both immunosuppressive agents produced a significant increase in mortality, this treatment had differing effects on the pathological involvement of gray and white-matter structures in the central nervous system. The central nervous system of immunosuppressed TMEV-infected mice had increased microglial cell proliferation and neuronal necrosis, longer maintenance of high virus levels and spread of virus antigen to involve the neocortex and hippocampal complex. These observations indicate that TMEV causes a cytolotic infection of neurons and possibly other cells in gray matter. In contrast, immunosuppression produced a dramatic reduction in mononuclear inflammatory cells in the leptomeninges and spinal cord white matter of infected mice and prevented demyelination. Further, virus antigen was not detected in the leptomeninges and white matter of immunosuppressed and infected mice. These findings suggest that demyelination of TMEV infection is immune mediated.     

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. II. Survey of host immune responses and central nervous system virus titers in inbred mouse strains

Previous studies using mouse strains with limited genetic differences and H-2 haplotypes demonstrated that susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease strongly correlated with chronically high levels of TMEV-specific delayed-type hypersensitivity (DTH), but not with TMEV-specific T cell proliferation (Tprlf), serum antibody responses, or with CNS virus titers. To determine if this correlation would be supported by analysis of these parameters in a more thorough genetic survey, ten inbred mouse strains, representing a wide variety of genetic backgrounds and H-2 haplotypes, were inoculated intracerebrally (i.c.) with the BeAn strain of TMEV. Significant TMEV-specific DTH was observed in all highly susceptible strains, but was not detectable in intermediate and resistant strains. TMEV-specific serum antibody titers also appeared to correlate with susceptibility to demyelinating disease, however even resistant strains had high antibody responses. Significant differences in CNS TMEV titers existed between strains, but did not correlate with disease susceptibility. DTH and Tprlf responses were observed in 3/4 resistant strains following peripheral immunization with UV-inactivated TMEV indicating that most resistant strains are genetically capable of mounting virus-specific cell-mediated immune (CMI) responses. The data extends our knowledge of host immune responses and virus titers in many different inbred mouse strains persistently infected with TMEV, supports the hypothesis that the demyelination in highly susceptible mice involves a TMEV-specific DTH response, and suggests that the genetic ability to mount specific DTH responses is necessary, but not sufficient for development of the demyelinating disease.     

Inhibition of Theiler's virus-induced demyelination in vivo by tumor necrosis factor alpha

Employing a murine model of multiple sclerosis which utilizes intracranial injection of Theiler's virus murine encephalomyelitis (TMEV) into SJL/J mice, we tested the potential role of tumor necrosis factor alpha (TNF-alpha) in ameliorating CNS demyelination. Infection with TMEV caused early grey matter inflammation (7 days post-infection) in the brain and spinal cord followed by chronic demyelination (35 days post-infection) in the spinal cord. Administration of recombinant human or mouse TNF-alpha starting 12 h prior to infection and then three times weekly had minimal effect on development of grey matter inflammation in the spinal cord. In contrast, TNF-alpha dramatically reduced demyelination present in spinal cord on days 14 and 35 after TMEV infection (P less than 0.01) when compared to controls. CNS virus titers of TMEV were not modified by TNF-alpha administration as measured on days 7, 14, and 35 following infection. In vivo administration of TNF-alpha inhibits TMEV-induced demyelination in susceptible SJL/J mice without affecting virus replication in the CNS.     

Theiler's virus-induced demyelination in mice immunosuppressed with anti-IgM and in mice expressing the xid gene

Intracerebral infection with Theiler's murine encephalomyelitis virus produces chronic immune-mediated demyelination in susceptible strains of mice. We examined the role of Ig in the pathogenesis of demyelination. In susceptible SJL/J mice (H-2s), suppression of B cell responses with IgG fraction of goat anti-mu (anti-mu IgG) from birth resulted in increased numbers and severity of demyelinating lesions in the spinal cord 35 days after infection. In contrast, treatment of resistant C57BL/10 (H-2b), C57BL/6 (H-2b), or B10.D2 (H-2d) mice with anti-mu IgG had no apparent effect since these mice did not develop demyelination or inflammation in the spinal cord following infection. Similar results were obtained with certain strains of B-cell deficient mice that exhibit the xid gene mutation. Male CBA/NJ (xid) showed increased meningeal inflammation and demyelination compared to male CBA/J mice. However, B6.CBAN, C3.CBAN, or C.CBAn mice showed no or minimal evidence of demyelination despite the presence of the xid mutation. In the SJL/J mouse, the majority of the humoral immune response to virus antigen was restricted to the IgG2b and IgM isotypes. These data indirectly support the hypothesis that immunoglobulins protect partially against development of virus-induced demyelination in susceptible but not resistant animals. In addition, the data argue strongly against the hypothesis that TMEV-induced demyelination is mediated predominantly by humoral autoimmune or humoral viral immune mechanisms.     

Role of T cells in resistance to Theiler's virus infection.

Intracerebral infection of C57BL/10SNJ mice with Theiler's virus results in acute encephalitis with subsequent virus clearance and absence of spinal cord demyelination. In contrast, infection of SJL/J mice results in acute encephalitis, virus persistence, and immune-mediated demyelination. These experiments examined the role of T-cell subsets in the in vivo immune response to Theiler's virus in resistant C57BL/10SNJ mice. Depletion of T-cell subsets with monoclonal antibodies (mAbs) directed at CD3 (pan-T-cell marker), CD4+ (class II-restricted) or CD8+ (class I-restricted) T cells resulted in increased frequency of paralysis and death as a result of acute encephalitis. Neuropathologic studies 10 days after infection demonstrated prominent necrosis, primarily in the pyramidal layer of hippocampus and in the thalamus of mice depleted of T-cell subsets. In immunosuppressed and infected C57BL/10SNJ mice, analysis of spinal cord sections 35 days after infection demonstrated small demyelinated lesions relatively devoid of inflammatory cells even though virus antigen could be detected by immunocytochemistry. Both CD4+ and CD8+ T cells are important in the resistance to infection with Theiler's virus in C57BL/10SNJ mice. However, subsequent spinal cord demyelination, to the extent observed in susceptible mice, depends on the presence of virus antigen persistence and a competent cellular immune response.     

V(beta)8(+) T cells protect from demyelinating disease in a viral model of multiple sclerosis

Previous studies illustrated the influence of T cell subsets on susceptibility or resistance to demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. Genetic segregation analysis showed a correlation with disease phenotype in this model with particular V(beta) genes. In this study we investigated the contribution of specific V(beta) TCR to the pathogenesis of virus-induced demyelinating disease. Spectratype analysis of cells infiltrating the CNS early in infection demonstrated an over-representation of V(beta)8(+) T cells in mice expressing a susceptible H-2 haplotype. We infected transgenic mice expressing the V(beta)8.2 TCR directed against a non-TMEV antigen and found an increase in demyelinating disease in mice of either susceptible or resistant background compared with littermate controls. In addition, depletion studies with an anti-V(beta)8-specific antibody in both susceptible (B10.Q) and resistant (C57BL/6) mice resulted in increased demyelination. TCR analysis of VP2-specific cytotoxic T cell clones from mice with a resistant genotype identified only the V(beta)8.1 TCR, suggesting that limited T cell diversity is critical to TMEV clearance. Together, these results support a protective role for V(beta)8(+) T cells in virus-induced demyelinating disease.     

CD40/CD40L interaction is essential for the induction of EAE in the absence of CD28-mediated co-stimulation

CD28 provides a co-stimulatory signal critical for optimal T cell activation. We and others have shown that the B7/CD28 co-stimulatory pathway is a major regulatory pathway for the control of immune responses. Experimentally induced models of autoimmunity have been shown to be prevented or reduced in intensity in mice deficient for CD28. Here, we show that EAE and accompanying neuroantigen-specific immune responses are drastically reduced in the absence of CD28. However, we go on to show that EAE can be induced in CD28-deficient mice following two immunizations. After re-immunization, CD28-deficient mice develop severe EAE with myelin-specific responses equal to those of wildtype controls, and extensive demyelination in the spinal cord. Treatment of CD28-deficient mice with anti-CD40L at the time of immunization significantly reduced DTH responses and prevented the development of EAE following two immunizations, indicating a critical role for CD40/CD40L signaling in the absence of CD28. Taken together, our results indicate that CD28-mediated co-stimulation does not regulate immunological anergy. Instead, CD28 appears to adjust the threshold for activation and expansion of autoreactive cells.     

Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler's virus infection.

Mice experimentally infected with Theiler's murine encephalomyelitis virus (TMEV) develop a persistent infection of the central nervous system (CNS). The most striking feature of this infection is the occurrence of inflammatory primary demyelination in the spinal cord white matter. The pathogenesis of myelin degeneration in this model has not been clarified, but morphologic and immunologic data suggest that the host immune response plays a major role in the production of myelin injury. Because of low virus titers in infected adult mice and of the small size of TMEV, virus particles have never been observed in this demyelinating model. Yet elucidation of the types of cells in the CNS supporting virus replication would be important for a better understanding of both virus persistence and virus-induced demyelinating pathology. The present paper is a sequential study of the localization of TMEV in the spinal cord in infected mice by ultrastructural immunohistochemical techniques. Results indicate that virus replication is mainly in neurons during the acute phase of the disease, while in the chronic phase viral inclusions are mainly found in macrophages in and around demyelinating lesions. Other cells are also infected, but to a lesser degree. In the neuronal system both axoplasmic and dendritic flow appear to facilitate the spread of virus in the CNS. In macrophages, the presence of virus particles and the association of virus with altered components of the cytoskeleton support active virus production rather than simple internalization. The macrophage appears to play an important role in both the establishment of virus persistence and in the process of demyelination in this animal model.     

Initial capsid-specific CD4(+) T cell responses protect against Theiler's murine encephalomyelitisvirus-induced demyelinating disease

Central nervous system (CNS) infection by Theiler's murine encephalomyelitis virus (TMEV) causes an immune-mediated demyelinating disease similar to human multiple sclerosis in susceptible mice. To understand the pathogenic mechanisms, we analyzed the level, specificity, and function of CD4(+) Th cells in susceptible SJL/J and resistant C57BL/6 mice. Compared to resistant mice, susceptible mice have three- to fourfold higher levels of overall CNS-infiltrating CD4(+) T cells during acute infection. CD4(+) T cells in the CNS of both strains display various activation markers and produce high levels of IFN-gamma upon stimulation with anti-CD3 antibody. However, susceptible mice display significantly fewer (tenfold) IFN-gamma-producing Th1 cells specific for viral capsid epitopes as compared to resistant mice. Furthermore, preimmunization with capsid-epitope peptides significantly increased capsid-specific CD4(+) T cells in the CNS during the early stages of viral infection and delayed the development of demyelinating disease in SJL/J mice. This suggests a protective role of capsid-reactive Th cells during early viral infection. Therefore, a low level of the protective Th1 response to viral capsid proteins, in conjunction with Th1 responses to unknown epitopes may delay viral clearance in susceptible mice leading to pathogenesis of demyelination during acute infection, as compared to resistant mice.     

Oral administration of live virus protects susceptible mice from developing Theiler's virus-induced demyelinating disease

Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in an immune-mediated demyelinating disease similar to human multiple sclerosis. TMEV infection is widely spread via fecal-oral routes among wild mouse populations, yet these infected mice rarely develop clinical disease. Oral vaccination has often been used to protect the host against many different infectious agents, although the underlying protective mechanism of prior oral exposure is still unknown. To understand the mechanisms involved in protection from demyelinating disease following previous oral infection, immune parameters and disease progression of mice perorally infected with TMEV were compared with those of mice immunized intraperitoneally following intracerebral infection. Mice infected perorally, but not intraperitoneally, prior to CNS viral infection showed lower chronic viral persistence in the CNS and reduced TMEV-induced demyelinating disease. However, a prolonged period of post-oral infection was necessary for effective protection. Mice orally pre-exposed to the virus displayed markedly elevated levels of antibody response to TMEV in the serum, although T cell responses to TMEV in the periphery were not significantly different between perorally and intraperitoneally immunized mice. In addition, orally vaccinated mice showed higher levels of early CNS-infiltration of B cells producing anti-TMEV antibody as well as virus-specific CD4(+) and CD8(+) T cells in the CNS compared to intraperitoneally immunized mice. Therefore, the generation of a sufficient level of protective immune responses appears to require a prolonged time period to confer protection from TMEV-induced demyelinating disease.     

The relationship between viral RNA, myelin-specific mRNAs, and demyelination in central nervous system disease during Theiler's virus infection.

The DA strain of Theiler's murine encephalomyelitis virus (DAV) causes a chronic demyelinating disease in susceptible mouse strains. To elucidate the pathogenesis of DAV-induced demyelination, the authors investigated the spatial and chronologic relationship between virus (antigen and RNA), myelin-specific mRNAs, and demyelination in DAV-infected mice using immunohistochemistry, in situ hybridization, and slot blot hybridization analyses. In spinal cord white matter, viral RNA was detected easily in ventral root entry zones 1 to 2 weeks after infection. Viral RNA increased to maximum levels by 4 weeks after infection, which was associated with inflammation and mild demyelination. At 8 to 12 weeks after infection, when demyelination became most extensive, viral RNA was significantly decreased. Demyelination did not chronologically or spatially parallel the presence of viral RNA within the spinal cord. Decrease of myelin-specific mRNAs, including myelin-basic protein and proteolipid protein mRNAs, was observed within the demyelinating lesions with or without detectable viral RNA. These results indicate that a viral infection of white matter in the early phase of the infection initiates spinal cord disease leading to demyelination, but later an ongoing immunopathologic process contributes to the presence of extensive demyelination.     

Antibody response is required for protection from Theiler's virus-induced encephalitis in C57BL/6 mice in the absence of CD8+ T cells

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that beta2M-deficient C57BL/6 mice lacking functional CD8+ T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice (muMT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected muMT and control B6 mice, the levels of CD4(+) T cells were higher in the CNS of muMT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in muMT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated muMT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8+ T cells.     

Protective and Detrimental Roles for Regulatory T Cells in a Viral Model for Multiple Sclerosis

Multiple sclerosis (MS) has been proposed to be an immune‐mediated disease in the central nervous system (CNS) that can be triggered by virus infections. In Theiler's murine encephalomyelitis virus (TMEV) infection, during the first week (acute stage), mice develop polioencephalomyelitis. After 3 weeks (chronic stage), mice develop immune‐mediated demyelination with virus persistence, which has been used as a viral model for MS. Regulatory T cells (Tregs) can suppress inflammation, and have been suggested to be protective in immune‐mediated diseases, including MS. However, in virus‐induced inflammatory demyelination, although Tregs can suppress inflammation, preventing immune‐mediated pathology, Tregs may also suppress antiviral immune responses, leading to more active viral replication and/or persistence. To determine the role and potential translational usage of Tregs in MS, we treated TMEV‐infected mice with ex vivo generated induced Tregs (iTregs) on day 0 (early) or during the chronic stage (therapeutic). Early treatment worsened clinical signs during acute disease. The exacerbation of acute disease was associated with increased virus titers and decreased immune cell recruitment in the CNS. Therapeutic iTreg treatment reduced inflammatory demyelination during chronic disease. Immunologically, iTreg treatment increased interleukin‐10 production from B cells, CD4⁺ T cells and dendritic cells, which may contribute to the decreased CNS inflammation.     

Cytotoxic CD8+ T cell ablation enhances the capacity of regulatory T cells to delay viral elimination in Theiler's murine encephalomyelitis

Theiler's murine encephalomyelitis (TME) of susceptible mouse strains is a commonly used infectious animal model for multiple sclerosis. The study aim was to test the hypothesis whether cytotoxic T cell responses account for the limited impact of regulatory T cells on antiviral immunity in TME virus‐induced demyelinating disease (TMEV‐IDD) resistant C57BL/6 mice. TME virus‐infected C57BL/6 mice were treated with (i) interleukin‐2/‐anti‐interleukin‐2‐antibody‐complexes to expand regulatory T cells (“Treg‐expansion”), (ii) anti‐CD8‐antibodies to deplete cytotoxic T cells (“CD8‐depletion”) or (iii) with a combination of Treg‐expansion and CD8‐depletion (“combined treatment”) prior to infection. Results showed that “combined treatment”, but neither sole “Treg‐expansion” nor “CD8‐depletion,” leads to sustained hippocampal infection and virus spread to the spinal cord in C57BL/6 mice. Prolonged infection reduces myelin basic protein expression in the spinal cord together with increased accumulation of β‐amyloid precursor protein in axons, characteristic of myelin loss and axonal damage, respectively. Chronic spinal cord infection upon “combined treatment” was also associated with increased T and B cell recruitment, accumulation of CD107b⁺ microglia/macrophages and enhanced mRNA expression of interleukin (IL)‐1α, IL‐10 and tumor necrosis factor α. In conclusion, data revealed that the suppressive capacity of Treg on viral elimination is efficiently boosted by CD8‐depletion, which renders C57BL/6 mice susceptible to develop chronic neuroinfection and TMEV‐IDD.