Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism

Abstract

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.

FIG. 1.

EGFP-tagged isogenic recombinant viruses differ in the spike gene. A schematic diagram of the replacement of gene 4a and part of gene 4b in the MHV genome expressing EGFP. SJHM-RA59_EGFP expresses the JHM spike gene, RA59_EGFP expresses the A59 spike gene, and SMHV2-RA59_EGFP expresses the MHV-2 spike gene in the background of the A59 gene. All three recombinant viruses derived are isogenic, differing only in the spike gene.

FIG. 2.

CNS pathology of EGFP-expressing MHVs. The upper panel shows the observed demyelination. Luxol fast blue stained spinal cord sections of mice infected with SJHM-RA59_EGFP, RA59_EGFP, or SMHV2-RA59_EGFP, at day 30 postinfection. Thin arrows indicate a region of demyelination, and arrowheads indicate normal myelinated area of white matter. Original magnification, ×20. The middle panel shows the observed encephalitis. The microglial nodules shown are from H&E-stained basal forebrain sections of mice infected with EGFP-tagged MHVs at day 7 postinfection. Arrows indicate the microglial nodules (microglia with elongated nuclei) and lymphocytes in the vicinity of neurons in mice infected with SJHM-RA59_EGFP, RA59_EGFP, and SMHV2-RA59_EGFP. Original magnification, ×200. The lower panel shows the observed meningitis. H&E-stained sections from the basal forebrain of mice infected with EGFP-tagged MHVs at day 7 postinfection; shown are SJHM-RA59_EGFP, RA59_EGFP, and SMHV2-RA59_EGFP. In all cases, there is a brisk leptomeningeal lymphocytic inflammatory infiltrate. Original magnification, ×200.

FIG. 3.

Identification of EGFP MHV-infected neurons in the brain at day 5 postinfection. Mice were infected intracranially with SJHM-RA59_EGFP, RA59_EGFP, and SMHV2-RA59_EGFP. Infected mice were sacrificed at day 5 postinfection, and the brains were processed, sectioned, and then labeled with anti-MAP2b as primary antibody and with Texas red goat anti-mouse IgG as secondary antibody. EGFP fluorescence (green) was used to detect viral antigen-positive cells in the basal forebrain. Red fluorescence shows the corresponding labeling of MAP2b in neurons. Merged images show the colocalization of MAP2b- and EGFP-positive cells. Arrows identify cells double positive for viral antigen and MAP2b.

FIG. 4.

Colocalization of EGFP-positive cells with astrocyte marker at day 5 postinfection. Mice were infected intracranially with SJHM-RA59_EGFP, RA59_EGFP, and SMHV2-RA59_EGFP. Infected mice were sacrificed at day 5 postinfection; brains were processed, sectioned, and then immune-labeled with anti-GFAP (astrocytic marker) antiserum, and then stained with Texas red goat anti-rabbit IgG as secondary antibodies. EGFP fluorescence was used to detect viral antigen-positive cells; red fluorescence was used to detect GFAP in astrocytes. Merged images show the colocalization of GFAP- and EGFP-positive cells. Arrows identify cells double positive for viral antigen and GFAP.

FIG. 5.

Viral antigen expression in spinal cord sections of mice infected with EGFP-expressing viruses. Mice were inoculated intracranially with SJHM-RA59_EGFP, RA59_EGFP, and SMHV2-RA59_EGFP. Mice were sacrificed at days 5 and 7 postinfection, and spinal cord sections were stained with anti-nucleocapsid antiserum. Representative hemisections and complete sections of spinal cords are shown from the cervical regions of the spinal cords of mice infected on day 5 (upper panel) and day 7 (middle panel). In SMHV2-RA59_EGFP-infected mice, occasional viral antigen distribution in the white matter and gray-white matter junction is shown in the lower panel.