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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.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.

This study included 49 patients with clinical multiple sclerosis (MS), 105 patients with other neurological diseases (OND), and 30 controls. It compared seven assays for CNS IgG synthesis with the oligoclonal banding (agarose electrophoresis) method. A newly developed assay which determined the differences between the measured and calculated CSF/serum IgG ratio (M-C value), using albumin as a reference protein, was particularly sensitive to the diagnosis of MS. In 40/46 (87%) of patients with MS, the M-C value was 0.001 or more, while oligoclonal banding was found in CSF of 38/49 (78%). In the 30 controls, the M-C value was invariably less than 0.001 and oligoclonal banding was not found. In patients with OND, 26/104 (25%) had an M-C value of 0.001 of greater while 11/105 (11%) had oligoclonal banding in CSF. The M-C value also offers a convenient means of quantifying CNS IgG synthesis during disease activity of treatment. It is concluded that the combined use of the oligoclonal banding method and the M-C value determination gives the greatest predictive value for the diagnosis of MS.

Murine coronaviruses undergo RNA recombination at a very high frequency. We have obtained a series of recombinant viruses using neutralizing monoclonal antibodies in conjunction with temperature-sensitive markers. All of the recombinants obtained have a crossover within gene C, which encodes the peplomer protein of the virus. The genetic structure of these recombinants suggests that the antigenic regions recognized by these neutralizing monoclonal antibodies are localized on the carboxyl-terminal one-third of the peplomer protein. Since the two monoclonal antibodies used are also associated with the critical determinants of virus neuropathogenicity, we conclude that both the neutralizing antibody binding sites and determinants of pathogenicity are localized at the carboxyl-terminal one-third of the peplomer. The variation of crossover sites in different recombinant viruses also allowed precise mapping of additional antigenic sites. RNA recombination thus presents a powerful genetic tool, and the carboxyl-terminal localization of the biological functions of peplomers suggests a distinct conformation of these viral membrane proteins.

A small plaque variant of JHM virus has a markedly reduced ability to kill mice following intracerebral inoculation. Spinal cords of mice surviving 13 to 16 months following acute infection with this variant were examined ultrastructurally. Multiple subpial areas of demyelination in the anterior and lateral white matter were found in five of 13 mice. The lesions had more gliosis, fewer oligodendrocytes, and less remyelination than has been described following other infections with JHM virus. No conclusive evidence of active demyelination or viral-like particles was found. The pathogenesis of the lesions observed may be due to a persistent, attenuated infection of oligodendrocytes or to immunologic processes. These lesions were similar to chronic multiple sclerosis plaques. Therefore, this variant should prove to be a useful tool for studying the long-term effects of viral-induced demyelinating diseases.

We have investigated an enzyme-linked immunosorbent assay (ELISA) for mouse IgG using affinity-purified goat anti-mouse antibodies for capture and detection. This assay was used to measure the absolute or weight/volume concentration of murine monoclonal antibody in hybridoma supernatants. Bovine or subclasses except IgG3 in the 1-20 ng/ml range. Antibody capture was essentially complete in the optimized assay. In combination with an antigen-dependent ELISA, the assay allowed estimation of the absolute concentration of specific monoclonal antibody in ascites. These rapid and relatively simple assays may be applicable in many situations in which a practical means of measuring murine monoclonal antibodies in weight/volume units is needed.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.

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.