1987;68:1659C1667. no deficits despite demyelination. In all strains, deletion of CD4 but not CD8 resulted in a decreased delayed-type hypersensitivity response to viral antigen. We conclude that each T-cell subset makes a discrete and nonredundant contribution to protection from viral persistence and demyelination in Purvalanol A resistant strains. In contrast, in susceptible strains, CD8+ T cells do not provide protection against chronic demyelinating disease. Furthermore, in persistent TMEV infection of the central nervous system, neurologic deficits appear to result either from the absence of a protective class II-restricted immune response or from the presence of a pathogenic class I-restricted response. Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system (CNS) in humans. MS lesions are characterized by foci of inflammation, myelin destruction, and formation of astrocytic scars known as plaques. The presence of CD4+ T cells, CD8+ T cells (11), and macrophages in lesions suggests that pathogenesis is immunologically mediated; however, the specific contribution of specific cell types remains unknown (12, 44, 45). Flt4 Although the etiology of MS is unknown, virus infection is the only epidemiological factor consistently associated with clinical exacerbation (43), and beta interferon, a cytokine with multiple known antiviral properties (46), is the only therapeutic agent definitively shown to decrease exacerbation and limit disability in MS (46). Therefore, the study of viral models of demyelination is extremely relevant. Theilers murine encephalomyelitis virus (TMEV), a picornavirus, induces a pathological and clinical disease similar to MS (24). Intracerebral infection with the Daniel strain (DA) of TMEV induces transient, acute neuronal polioencephalitis followed by chronic white matter demyelination and neurologic deficits in mice with susceptible ( 0.05 by Students test and is specified in the text. Virus plaque assays. Viral Purvalanol A titers in clarified CNS homogenates were determined by plaque assay as described previously (36). On days 7 and 45 after infection, CNS homogenates Purvalanol A were prepared from brains and spinal cords that had been removed aseptically. A 10% (wt/vol) homogenate was prepared in Dulbecco modified Eagle medium, sonicated three times for 20 s each, and clarified by centrifugation. Virus preparations were stored at ?70C before use. Each assay was performed at least in duplicate, and in most cases in triplicate, on coded samples without knowledge of experimental groups. Data were expressed as log10 PFU per gram of CNS tissue. Statistical significance is reported at 0.05 by the Mann-Whitney rank sum test and is specified in the text. Immunocytochemistry for virus antigen. For immunoperoxidase studies, coronal spinal cord sections (five or six per mouse) from perfused animals were stored in 0.1 M phosphate buffer, rinsed in 0.1 M Tris buffer with 25 mM hydroxylamine (pH 7.4), treated with 10% dimethyl sulfoxide for 1 h, and quick-frozen in liquid nitrogen-chilled isopentane. Cryostat sections were incubated with a polyclonal rabbit antiserum to purified TMEV DA virions (36), which specifically reacts to all structural proteins of TMEV (36). Slides were developed by using the avidin-biotin immunoperoxidase system (Vector Laboratories, Burlingame, Calif.). For quantitative analysis, a Zeiss microscope attached to a camera lucida was used to project the spinal cord images onto a ZIDAS (Carl Zeiss Inc., Oberkochen, Germany) digitizing tablet. Spinal cord areas were traced to determine the total area (expressed in square millimeters). We analyzed a minimum of 5.89 mm2 to a maximum of 28.07 mm2 of spinal cord for each mouse. The total area of spinal cord examined by combining all animals from the experimental groups was 931 mm2. The number of virus antigen-positive cells for each mouse was counted and expressed Purvalanol A per area of spinal cord. In situ hybridization. In situ hybridization for TMEV RNA was carried out as described previously (20). Briefly, fixed sections were treated with 1 g of proteinase K per ml, acetylated, and prehybridized for 4 h at room temperature with buffer containing deionized formamide, Denhardts solution, sodium chloride, salmon sperm DNA, and yeast tRNA. Slides were hybridized with 35S-labeled 253-bp (nucleotides 3053 to 3305) and 363-bp (nucleotides 3306 to 3668) cDNA probes corresponding to VP1 of TMEV DA (22). The cDNA probes were obtained by double digestion of the VP1 plasmid with 0.01 by Students test). In contrast,.