The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM ("receptor-independent spread"). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.Mouse hepatitis virus (MHV) strain JHM is an enveloped, nonsegmented positive-strand RNA virus belonging to the Coronaviridae family within the Nidovirales order (7). JHM infection of experimental animals has been extensively used to study virus pathogenesis in the central nervous system (CNS). Susceptible mice develop an acute, fatal viral encephalomyelitis after intranasal or intracranial inoculation, with clinical signs that include hunching, ruffled fur, irritability, and lethargy. Viral antigen is detected in gray-and white-matter structures throughout the brain. Rodents chronically infected with MHV develop demyelinating diseases with many similarities to multiple sclerosis in humans (21,55,56). Chronic infection results if animals are infected with an attenuated virus or with a virulent virus in the presence of anti-JHM antibodies or T cells (5,13,26,31,39,48,57,69). In one model of chronic infection, suckling mice were infected with JHM and nursed by dams previously immunized against the virus (48). Under these conditions, sufficient antibody was transmitted to suckling mice to prevent acute encephalitis but not to mediate virus clearance. Consequently, 40 to 90% of mice developed hindlimb paralysis and histological evidence of a demyelinating encephalomyelitis at 3 to 8 weeks postinfection (p.i.) (48). Since the inception of this maternal antibody model, we have used a specific strain of JHM, JHM.Iowa (JHM.IA), for all of our experiments.

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs). Microglia also express CD11c during this phase of the infection. CD11c(+) macrophages in the infected CNS exhibit variable properties of immature antigen-presenting cells (APCs), with modestly increased CD40 and MHC expression, and equivalent potent antigen uptake when compared with CD11c(-) macrophages. Furthermore, CDllc(+) and F4/80(+) macrophages and microglia are localized to areas of demyelination, in some instances directly associated with damaged axons. These results suggest that chronic CNS infection results in the appearance of CD11c-expressing macrophages from the blood that exhibit properties of immature APCs, are closely associated with areas of demyelination, and may act as final effectors of myelin destruction.

Mouse hepatitis virus strain JHM causes a chronic demyelinating disease in susceptible strains of rodents. Demyelination does not develop in infected RAG1؊/؊ (recombination activation gene-deficient) mice but can be induced by several experimental interventions, including adoptive transfer of virus-specific T cells or antibodies. A common feature of demyelination in these models is extensive infiltration of macrophages/ microglia into the white matter. The data obtained thus far do not indicate whether macrophage/microglia infiltration, in the absence of T cells or antibody, is sufficient to mediate demyelination. To determine whether the expression of a single macrophage chemoattractant, in the context of virus infection, could initiate the demyelinating process, we engineered a recombinant coronavirus that expressed the chemokine CCL2/monocyte chemoattractant protein-1. CCL2 has been implicated in macrophage infiltration into the central nervous system and is involved in demyelination in many experimental models of demyelination. Extensive macrophage/microglia infiltration and demyelination has developed in RAG1 ؊/؊ mice infected with this recombinant virus. Thus, these results suggest that the minimal requirement for demyelination is increased expression of a single macrophage-attracting chemokine in the context of an inflammatory milieu, such as that induced by a viral infection.

CTL escape mutations have been identified in several chronic infections, including mice infected with mouse hepatitis virus strain JHM. One outstanding question in understanding CTL escape is whether a CD8 T cell response to two or more immunodominant CTL epitopes would prevent CTL escape. Although CTL escape at multiple epitopes seems intuitively unlikely, CTL escape at multiple CD8 T cell epitopes has been documented in some chronically infected individual animals. To resolve this apparent contradiction, we engineered a recombinant variant of JHM that expressed the well-characterized gp33 epitope of lymphocytic choriomeningitis virus, an epitope with high functional avidity. The results show that the presence of a host response to this second epitope protected mice against CTL escape at the immunodominant JHM-specific CD8 T cell epitope, the persistence of infectious virus, and the development of clinical disease.

The human disease multiple sclerosis (MS) is an immunemediated, chronic inflammatory disease manifested clinically by neurological deficits and histologically by multiple foci of demyelination. T cells are detected in active demyelinating lesions and a critical role for these cells in demyelination has been clearly demonstrated in several animal models of demyelination, including rodents with experimental autoimmune encephalitis (EAE) and mice infected with coronaviruses or Theiler's murine encephalomyelitis virus. [1][2][3] Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHM) develop acute and chronic demyelinating diseases. We and others 4 -6 have shown that demyelination was not detected in JHM-infected mice lacking T and B cells [either mice with severe combined immunodeficiency or mice lacking functional recombination activating enzyme 1 (RAG1 Ϫ/Ϫ )]. However, adoptive transfer of syngeneic splenocytes from JHM-immune mice resulted in rapid and reproducible demyelination. 6,7 Depletion of T cells abrogated demyelination showing that T cells were necessary and B or other splenic cells were not sufficient for demyelination to occur. Either CD8 or CD4 T cells, in the absence of the other subset, were able to mediate demyelination in this model. 4 In these experiments, T cells were transferred into RAG1 Ϫ/Ϫ mice 4 days after they were immunized with JHM. The innate immune system was activated by JHM infection before Tcell transfer, as shown by up-regulated expression of several proinflammatory cytokines and chemokines, such as tumor necrosis factor-␣, MIP-2, CCL7 (MCP-3), CCL4 (MIP-1␤), CCL2 (MCP-1), CXCR10 (IP-10), and CCL5 (RANTES) in the central nervous system (CNS). 8 This intense inflammatory milieu is likely critical for the rapid recruitment and activation of T cells to the CNS after adoptive transfer.Less is known about the role of humoral immune factors in MS, but several features suggest that B cells or antibodies are involved in myelin destruction. 9 Oligoclonal expansion of B cells is observed in the cerebrospinal fluid of patients with MS. Also, high levels of immunoglobulin are detected in the cerebrospinal fluid. 10 Some of these cerebrospinal fluid-derived antibodies are directed against myelin proteins and pathogens such as Epstein-Barr virus 11 and varicella-zoster virus.