John O. Fleming

Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated

Abstract The neurotropic mouse hepatitis viruses (MHV), in particular strain JHM (JHMV or MHV-4), cause experimental central nervous system demyelination that pathologically resembles multiple sclerosis, an important human demyelinating disease. The mechanism of JHMV-induced demyelination remains unclear, though its tropism for oligodendrocytes had led to the belief that JHMV causes demyelination by direct lysis of these myelin-producing cells. However, several studies have also implicated the involvement of immune responses in the demyelinating process. In this communication, we present evidence that generalized immunosuppression with gamma irradiation prevents JHMV-induced demyelination, a finding that was not limited to a particular strain of JHMV or to one strain of mouse. In addition, significant paralytic-demyelinating disease was restored to infected, irradiated mice after the adoptive transfer of nylon wool nonadherent splenic cells and appeared to be restricted by the major histocompatibility complex (MHC). These observations indicate that the principal mechanisms of JHMV-induced demyelination are most likely immunopathological.

Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus

Abstract Monoclonal antibodies were produced to JHMV-DL, a neurotropic member of the mouse hepatitis virus (MHV) or murine coronavirus group. Of 23 antibodies isolated, 10 were specific for the major envelope glycoprotein, gp180/90, 10 for the nucleocapsid protein, pp60, and 3 for the minor envelope glycoprotein, gp25. Eleven different MHV isolates were used in antibody binding assays to study antigenic relationships among the viruses. Each MHV isolate tested had a unique pattern of antibody binding, indicating that each is a distinct strain. Conservation of JHMV-DL antigenic determinants varied among the three proteins, with pp60 showing intermediate conservation, gp180/90 little conservation, and gp25 marked conservation in the different MHV strains. Monoclonal antibodies to pp60 proved most useful in delineating antigenic relationships among MHV strains. These antigenic groups correlated with pathogenic types, indicating that pp60 may be one of the gene products which mediates the distinct disease patterns manifested by different murine coronaviruses.

Monoclonal antibodies to the matrix (El) glycoprotein of mouse hepatitis virus protect mice from encephalitis

Abstract Monoclonal antibodies to the matrix or El glycoprotein of mouse hepatitis virus (MHV) were tested for their ability to protect mice from a normally lethal challenge of MHV-4. Four antibodies were tested, and two of these,J.1.3 and J.3.9, were protective. Protection did not correlate with virus neutralization in vitro, antibody isotype, recognition of a unique El antigenic site, or dependence on complement in vivo. Survival from acute encephalitis was followed by subacute demyelination, as has been shown with protection mediated by neutralizing monoclonal antibodies against the major glycoprotein, E2. These results demonstrate that antibodies which are specific for a viral matrix protein are able to alter the course of disease.

Sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity.

Abstract Mouse hepatitis virus (MHV), a coronavirus, causes encephalitis and demyelination in susceptible rodents. Previous investigations have shown that the MHV spike (S) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. To understand the molecular basis of MHV neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the JHM strain of MHV, which were selected with monoclonal antibodies (MAbs) specific for the S protein. We found that variant 2.2-V-1, which was selected with MAb J.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (NT) 3340, as compared to the parental JHM virus, which predominantly caused encephalitis. This site was in the S2 subunit of the S protein. In contrast, variant 7.2-V-1, which was selected with MAb J.7.2 and primarily caused encephalitis, had two point mutations at NT 1766 and 1950, which were in the S1 subunit. Finally, the double mutant 2.2/7.2-V-2, which was selected with both MAbs J.2.2 and J.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from NT 1523 to 1624 in the S1 and a point mutation at NT 3340 in the S2. We conclude that at least two regions of the S protein contribute to neuropathogenicity of MHV. We have also isolated a partial revertant of 2.2-V-1, which was partially resistant to MAb 1.2.2 but retained the same neuropathogenicity as the variant 2.2-V-1. This revenant retained the mutation at NT 3340, but had a second-site mutation at NT 1994, further confirming that NT 3340 contributed to the pathogenic phenotype of MHV. By comparing these results with MHV variants isolated in other laboratories, which had mutations in other sites on the S gene and yet retained the demyelinating ability, we suggest that the ability of JHM viruses to induce demyelination is determined by the interaction of multiple sites on the S gene, rather than the characteristics of a single, unique site. Our study also revealed the possible presence of microheterogeneity of S gene sequence, particularly in the S1 region, in these viruses. The sequence microheterogeneity may also contribute to the differences in their biological properties.

Experimental demyelination induced by coronavirus JHM (MHV-4): molecular identification of a viral determinant of paralytic disease

Abstract The molecular basis for demyelination induced by the neurotropic murine coronavirus JHM (JHMV or MHV4) is unknown. We have attempted to explore this issue by using neutralizing monoclonal antibodies specific for the major JHMV glycoprotein (E2) to select sets of neutralization resistant (NR) antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 bind to topographically distinct sites on E2. NR variants selected with J.7.2, like parental JHMV, predominantly cause a fatal encephalitis when given intracerebrally to mice, while J.2.2-selected NR variants cause a subacute disease characterized by paralysis and severe demyelination. We report here that consecutive selection with both J.2.2 and J.7.2 monoclonal antibodies results in NR variants which are markedly attenuated in both encephalitic potential and ability to induce demyelination. Analysis of the different variants suggests that the subregion of E2 bound by monoclonal antibody J.7.2 may be a critical viral determinant of paralysis and demyelination in this model system.

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein

Abstract The synthesis and processing of the nucleocapsid protein (pp60) of the JHM strain of murine coronaviruses were examined. Pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). Immunoprecipitation using mouse anti-JHMV antiserum indicated that p57 was virus specific. Immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. The p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein.

Improvements in obtaining and characterizing mouse cerebrospinal fluid: Application to mouse hepatitis virus-induced encephalomyelitis

Abstract This report describes advances in techniques for analyzing cellular and humoral immune components in the cerebrospinal fluid (CSF) of the mouse that are applicable to other laboratory animals. CSF studies undertaken during experimental infection of mice with JHM strain virus (JHMV) of mouse hepatitis virus are presented. A critical pitfall which can lead to erroneous or invalid results is contamination of the CSF by even minute quantities of blood. Means of avoiding this contamination are attention to anatomical reference points, the use of a micropipet, and prior intracardiac perfusion of animals with phosphate-buffered saline. Cells in the CSF were typed as either B, T, polymorphonuclear, or mononuclear cells by the combination of a microcytotoxicity assay and histologic stains. A radioimmunoassay (RIA) allowed quantification of antibodies to JHMV in the CSF and indicated the presence of intrathecal synthesis of antibody in chronically infected mice. The combined use of these sensitive methods makes possible CSF analysis in individual mice rather than in pooled groups.

Characterization of the Structural Proteins of the Murine Coronavirus Strain A59 Using Monoclonal Antibodies

Abstract Monoclonal antibodies reacting with the A59 strain of mouse hepatitis virus (MHV-A59) were characterized and those specific to the E2 major envelope glycoprotein were studied in detail. Antibodies were tested for their ability to neutralize viral infectivity (N+ characteristic) and prevent viral-induced cell-to-cell fusion (F+ characteristic). All four possible combinations of activities reflecting E2 functions were found, i.e., N+F+, N−F−, N+F−, and N−F+. In addition, competitive binding studies with these monoclonal antibodies revealed two nonoverlapping antigenic regions. The first region, designated A, was recognized by antibodies which included each of the four functional types. Region B was identified by a single monoclonal antibody with N−F− activities. The existence of antibodies which only neutralize virus or only block viral-induced fusion implies that the structures on E2 which serve as targets for neutralization and which induce fusion are not identical. The critical determinants for neutralization and fusion must be closely related topographically on E2 since both N+F− and N−F+ antibodies recognize the same antigenic region.

Pathogenic Characteristics of Neutralization-Resistant Variants of JHM Coronavirus (MHV-4)

One of the most interesting aspects of murine coronaviruses is their ability to cause model, experimental neurological diseases in animals (1–10). In this situation, disease obviously depends on complex interactons between virus, host, and environment. The initial focus of our group has been to consider the specifically viral determinants of disease. We have adopted this approach because the virus is the least complex element of this biological system. Hopefully, the elucidation of viral components which play critical roles during disease will provide a basis for more general explanations of pathogenesis, including consideration of the host and environment.

Relapsing encephalomyelitis following transfer of partial immunity to JHM virus

Abstract Mice infected with the JHM strain mouse hepatitis virus (JHMV) develop a fatal encephalomyelitis with evidence of demyelination. It has previously been shown that the adoptive transfer of 5 × 107 nylon wool adherent (NWA) spleen cells from immunized donors to lethally infected recipients clears virus from the central nervous system (CNS) and prevents demyelination. Adoptive transfer of a smaller number (1 × 107) of NWA spleen cells from immunized donors also protects from death but does not significantly alter virus replication in the CNS during the acute phase of the infection. Moreover, these mice develop a transient non-fatal encephalomyelitis which occurs approximately 3 weeks post-infection. This delayed encephalomyelitis is associated with a mononuclear cell infiltration into the CNS but little or no evidence of virus replication or increased viral antigen. A virus-specific delayed-type hypersensitivity (DTH) response precedes this delayed onset of disease by 24 to 48 h. Resolution of disease correlates with a selective and permanent suppression of the JHMV-specific DTH reactivity. In addition, no virus-specific DTH is detected following adoptive transfer of viral-specific DTH effectors derived from immunized donors. In contrast, these mice respond to a heterologous antigen, KLH, suggesting that the resolution of the encephalitis is accompanied by a profound suppression in viral-specific DTH response.