Cellular reservoirs for coronavirus infection of the brain in beta2-microglobulin knockout mice

Abstract

Mouse hepatitis virus (MHV) A59 infection which causes acute encephalitis, hepatitis, and chronic demyelination, is one of the experimental models for multiple sclerosis. Previous studies showed that lethal infection of beta2-microglobulin 'knockout' (beta2M(-/-)) mice required 500-fold less virus and viral clearance was delayed as compared to infection of immunocompetent C57Bl/6 (B6) mice. To investigate the mechanism of the increased susceptibility of beta2M(-/-) mice to MHV-A59, we studied organ pathology and the distribution of viral antigen and RNA during acute and chronic infection. A59-infected beta2M(-/-) mice were more susceptible to acute encephalitis and hepatitis, but did not have increased susceptibility to demyelination. Viral antigen and RNA distribution in the brain was increased in microglia, lymphocytes, and small vessel endothelial cells while the distribution in neurons and glia was similar in beta2M(-/-) mice and B6 mice. Acute hepatitis and thymus cortical hypoplasia in beta2M(-/-) mice were delayed in onset but pathologic changes in these organs were similar to those in B6 mice. The low rate of demyelination in beta2M(-/-) mice was consistent with the low dose of the virus given. A less neurotropic virus MHV-2, caused increased parenchymal inflammation in beta2M(-/-) mice, but without demyelination. Thus, CD8+ cells were important for viral clearance from endothelial cells, microglia and inflammatory cells, but not from neuronal and glial cells. In addition, CD8+ cells played a role in preventing the spread of encephalitis.

Fig. 1

A-D Immunohistochemical analysis of MHV-A59-infected β₂M(-/-) mouse brains. A Neuronal infection is exhibited by positive immunostaining for MHV antigen in the cytoplasm of parahippocampal neurons. Note the morphological stigmata of neurons: the large cell size, the characteristic oval nucleus with the prominent nucleolus, and the thick axonal and dendritic processes. × 400. B Viral antigen detected in perivascular lymphocytes (arrowheads). × 200. C Viral antigen detected in endothelial cells delineating cerebral small venules and capillaries (arrowheads). × 400. D Area of prominent rod-shaped microglial cell proliferation including many that stain positively for viral antigen. × 100. E-H Combined in situ hybridization for viral genome and GFAP immunohistochemical staining for astrocytes. E GFAP-negative neuronal cells expressing viral genome (purple). Small purple dots represent positive viral genome in cross-sectioned cell processes. × 400. F Uninfected reactive astrocytes which are negative for viral genome by in situ hybridization expressing abundant GFAP by immunohistochemistry. × 400. G Infected astrocytes (arrows) exhibiting both GFAP (by immunohistochemistry with GFAP antibodies) and viral genome (in situ hybridization with MHV-specific probes). × 400. H A neuron expressing viral genome by in situ hybridization (purple) next to a GFAP-positive astrocyte (brown). × 400.

Fig. 2

Pathologic changes in β₂M(-/-) mice infected IC with MHV-2. A Periventricular necrotizing encephalitis. HE. × 100. B Parenchymal brainstem encephalitis with marked perivascular inflammatory infiltration. HE. × 100.

Fig. 3

Viral titers in organs of MHV-2-infected β₂M(-/-) mice (A) compared to MHV-2-infected B6 mice (B). β₂M(-/-) mice were infected IC with 5 × 10³ PFU of virus and B6 mice were infected IC with 5 × 10⁵ PFU of virus then sacrificed at various intervals postinfection; organs were aseptically removed and viral titers were determined by plaque assay.