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The spike (S) glycoprotein of mouse hepatitis virus (MHV) is a major determinant of neurovirulence. Using targeted recombination we previously demonstrated that the S gene of the highly neurovirulent MHV-4 conferred a dramatic increase in neurovirulence to the mildly neurovirulent MHV-A59. To identify the genetic determinants of neurovirulence within the MHV-4 spike, we generated isogenic recombinant viruses containing various MHV-4/MHV-A59 chimeric spike genes, and studied their phenotypes in vivo. The MHV-4/MHV-A59 chimeric spike genes consisted of either reciprocal exchanges between the S1 and S2 spike subunits, or smaller exchanges specifically in the hypervariable region (HVR) of S1. The chimeric spike gene containing recombinants all exhibited efficient replication in vitro, yet many were severely attenuated for virulence in vivo. Furthermore, these attenuated recombinants exhibited decreased titers of infectious virus in the brain relative to the parental recombinant viruses containing the full-length MHV-4 or MHV-A59 spike genes. This is the first report that compares the neurovirulence and pathogenesis of isogenic viruses with defined alterations in the MHV spike protein. From these studies, it appears that the interactions of multiple regions of the MHV spike, including the HVR, act in concert to allow for efficient infection of and virulence in the murine central nervous system.

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study, we used murine coronavirus, mouse hepatitis virus (MHV), infection of the central nervous system and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication compared to wild-type mice. Mice lacking IL-1 signaling experienced elevated viral replication but similar survival compared to wild-type controls. In the absence of IL-18, mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αβ T cells. These data suggest that inflammasome signaling is largely protective during murine coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.

The mouse pregnancy-specific glycoprotein 16 (PSG16) has been reported to be an alternative receptor for mouse hepatitis virus (MHV), some strains of which cause encephalitis in mice lacking the canonical receptor CEACAM1a. The known isoforms of PSG16 are N-terminally truncated relative to other PSG family proteins and are expressed in neurons as well as in the placenta. We have cloned a novel full-length isoform of psg16 that is also expressed in the brain, placenta, and retina but, like the truncated form, lacks MHV receptor activity when expressed on 293T cells, suggesting that PSG16 does not mediate CEACAM1a-independent spread of MHV.

Murine coronavirus, mouse hepatitis virus (MHV), causes various diseases depending on the strain and route of inoculation. Both the JHM and A59 strains, when inoculated intracranially or intranasally, are neurovirulent. Comparison of the highly virulent JHM isolate, JHM.SD, with less virulent JHM isolates and with A59 has been used to determine the mechanisms and genes responsible for high neuropathogenicity of MHV. The focus of this review is on the contributions of viral spread, replication, and innate and adaptive immunity to MHV neuropathogenesis. JHM.SD spreads more quickly among neurons than less neurovirulent MHVs, and is able to spread in the absence of the canonical MHV receptor, CEACAM1a. The observation that JHM.SD infects more cells and expresses more antigen, but produces less infectious virus per cell than A59, implies that efficient replication is not always a correlate of high neurovirulence. This is likely due to the unstable nature of the JHM.SD spike protein (S). JHM.SD induces a generally protective innate immune response; however, the strong neutrophil response may be more pathogenic than protective. In addition, JHM.SD induces only a minimal T-cell response, whereas the strong T-cell response and the concomitant interferon-γ (IFN-γ) induced by the less neurovirulent A59 is protective. Differences in the S and nucleocapsid (N) proteins between A59 and JHM.SD contribute to JHM.SD neuropathogenicity. The hemmagglutinin-esterase (HE) protein may enhance neuropathogenicity of some MHV isolates, but is unlikely a major contributor to the high neuroviruence of JHM.SD. Further data suggest that neither the internal (I) protein nor nonstructural proteins ns4, and ns2 are significant contributors to neurovirulence.

Infection of mice with mouse hepatitis virus (MHV) strain JHM (RJHM) induces lethal encephalitis, with high macrophage and neutrophil, but minimal T-cell, infiltration into the brain when compared to the neuroattenuated strain RA59. To determine if chemokine expression corresponds with the cellular infiltrate, chemokine protein and RNA levels from the brains of infected mice were quantified. RJHM-infected mice had lower T-cell (CXCL9, CXCL10), but higher macrophage-attracting (CCL2), chemokine proteins compared to RA59. RJHM also induced significantly higher CXCL2 (a neutrophil chemoattractant) mRNA compared to RA59. The neurovirulent spike gene chimera SJHM/RA59 induces high levels of T cells and macrophages in the brain compared to the attenuated SA59/RJHM chimera. Accordingly, SJHM/RA59 induced higher levels of CXCL9, CXCL10, and CCL2 protein compared to SA59/RJHM. Chemokine mRNA patterns were in general agreement. Thus, chemokine patterns correspond with the cellular infiltrate, and the spike protein influences levels of macrophage, but not T-cell, chemokines.

The cleavage and fusion properties of recombinant murine hepatitis viruses (MHV) were examined to assess the role of the cleavage signal in determining the extent of S protein cleavage, and the correlation between cleavage and induction of cell-to-cell fusion. Targeted recombination was used to introduce amino acid substitutions into the cleavage signal of the fusion glycoprotein (spike or S protein) of MHV strain A59. The recombinants were then used to address the question of the importance of S protein cleavage and viral-mediated cell-to-cell fusion on pathogenicity. Our data indicate that cleavage of spike is not solely determined by the amino acid sequence at the cleavage site, but may also depend on sequences removed from the cleavage site. In addition, efficient cell-to-cell fusion is not necessary for virulence.

Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent MHV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.

Murine hepatitis virus A59 infection of the central nervous system (CNS) results in CNS demyelination in susceptible strains of mice. In infected B-cell-deficient mice, demyelination not only occurred but was also more severe than in parental C57BL/6 animals. This increase may be due to the persistence of virus in the CNS in the absence of B cells. In mice lacking antibody receptors or complement pathway activity, virus did not persist yet demyelination was similar to parental mice. In infected RAG1(-/-) mice, moderately sized, typical demyelinating lesions were identified. Therefore, demyelination can occur in the absence of B and T cells.

Proteolytic processing of the mouse hepatitis virus strain A59 (MHV-A59) replicase gene product, pp1a, results in polypeptides p28, p65, p50, and p240 in infected cells. Based on previously identified p28 and p65 cleavage sites, a p50 cleavage site was proposed to occur between Ala-1262 and Ala-1263. Results of mutagenesis and in vitro cleavage assays show that PLP-1 was able to cleave in trans when the proposed p50 cleavage sequence replaced the p28 cleavage sequence. Mutagenesis was also used to investigate cleavage between Gly-904 and Val-905, a cleavage site predicted to produce a precursor of p65, p72, that was detected in cells infected with MHV strain JHM, but not with MHV-A59. No cleavage could be detected using substrate that carried both the p65 site and the predicted p72 cleavage sequence. Thus, it appeared that PLP-1 could recognize the proposed p50 sequence but not the predicted p72 site under the in vitro conditions used.

We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immunofluorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunofluorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immunofluorescent labeling of BHK cells expressing the MHV receptor (BHK(MHVR1)) and infected with MHV-A59 with a Golgi-specific anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies specific for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the first 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immunofluorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the first papain-like protease domain (PLP-1) was sufficient to associate this protein with the Golgi.

We examined murine hepatitis virus strain A59 (MHV-A59)-induced demyelinating disease in C57BL/6 mice which had previously been thymectomized at 25 days of age. Demyelination was observed in 51-96% of spinal cord quadrants examined 30 or 60 days post infection (dpi), indicating that neither an intact thymus nor thymic infection is a prerequisite to demyelination. Depletion of CD4+ or CD8+ T cells at 5, 7 or 10 dpi did not influence the extent of demyelination indicating that neither T cell subset is a major effector of demyelination. However, these findings do not exclude the possibility that T cells are involved in initiating demyelinating disease very early in infection.