B-cell-mediated lysis of cells infected with the neurotropic JHM strain of mouse hepatitis virus.

Cells expressing the spike (S) glycoprotein of the neurotropic JHM strain (JHMV) of mouse hepatitis virus (MHV) are susceptible to lysis by B cells derived from naïve mice, including B cells from perforin-deficient mice. Cytolysis requires interaction of the virus receptor and the viral S glycoprotein, is independent of other viral-induced components, and is not a unique property of B cells. Neutralizing anti-S-protein monoclonal antibodies (mAb) and a mAb specific for the viral receptor inhibit lysis. However, cells infected with an MHV strain unable to induce cell-cell fusion are resistant to lysis and lysis of JHMV-infected cells is inhibited by an anti-S-protein nonneutralizing mAb which prevents S-protein-mediated cell fusion. These data suggest that B cells may function as antibody-independent innate immune response during JHMV infection in vivo.

Viral induced demyelination.

Viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell-cell interactions and mechanisms of myelin destruction. They illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral-induced tissue damage. These studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (CNS). Although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (MS). This notion is supported by the persistent view that a viral infection acquired during adolescence might initiate MS after a long period of quiescence. Demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non-enveloped RNA and DNA viruses (Table 1). The mechanisms that ultimately result in the loss of CNS myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. Although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. This suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus-specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. Virus-induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own T cell compartment. Alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive T cells, i.e. molecular mimicry. Although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms.

Central nervous system expression of IL-10 inhibits autoimmune encephalomyelitis.

Multiple sclerosis, an inflammatory, demyelinating disease of the CNS currently lacks an effective therapy. We show here that CNS inflammation and clinical disease in experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis, could be prevented completely by a replication-defective adenovirus vector expressing the anti-inflammatory cytokine IL-10 (replication-deficient adenovirus expressing human IL-10), but only upon inoculation into the CNS where local infection and high IL-10 levels were achieved. High circulating levels of IL-10 produced by i. v. infection with replication-deficient adenovirus expressing human IL-10 was ineffective, although the immunological pathways for disease are initiated in the periphery in this disease model. In addition to this protective activity, intracranial injection of replication-deficient adenovirus expressing human IL-10 to mice with active disease blocked progression and accelerated disease remission. In a relapsing-remitting disease model, IL-10 gene transfer during remission prevented subsequent relapses. These data help explain the varying outcomes previously reported for systemic delivery of IL-10 in experimental autoimmune encephalomyelitis and show that, for optimum therapeutic activity, IL-10 must either access the CNS from the peripheral circulation or be delivered directly to it by strategies including the gene transfer described here.

Contributions of Fas-Fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system.

The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication.

Contributions of CD8+ T cells and viral spread to demyelinating disease.

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.

Microglia exhibit clonal variability in eliciting cytotoxic T lymphocyte responses independent of class I expression.

Microglia are important immunoregulatory cells within the central nervous system (CNS). Viral infection of primary microglia and splenic macrophage clones revealed that both exhibited a heterogeneous, but relatively low, sensitivity to cytolysis mediated by CD8(+) cytotoxic T lymphocytes (CTL). The majority of clones were poor in processing and presenting epitopes, despite triggering lysis when coated with peptide. These characteristics were retained by stable microglia lines. Reduced lysis did not correlate with class I expression and IFN-gamma treatment only partially enhanced recognition. In contrast, targeting the epitope into the endoplasmic reticulum restored cytolysis to levels achieved with exogenous peptide. An inherent resistance to cytolysis was revealed by efficient engagement of T cells in competition assays and the inability of saturating peptide to enhance cytolysis. These data suggest that microglia heterogeneity in antigen processing, in addition to low sensitivity to CTL lysis, contributes to limited CD8(+) T cell responses and viral CNS persistence.

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10.

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.

Mouse hepatitis virus strain JHM infects a human hepatocellular carcinoma cell line.

Mouse hepatitis virus (MHV) strain JHM is a coronavirus that causes encephalitis and demyelination in susceptible rodents. The known receptors for MHV are all members of the carcinoembryonic antigen family. Although human forms of the MHV receptor can function as MHV receptors in some assays, no human cell line has been identified that can support wild-type MHV infection. Here we describe the infection of a human hepatocellular carcinoma cell line, HuH-7, with MHV. HuH-7 cells were susceptible to strains JHM-DL and JHM-DS, yielding virus titers nearly identical to those seen in mouse DBT cells. In contrast, HuH-7 cells were only marginally susceptible or completely resistant to infection by other MHV strains, including A59. JHM produced a strong cytopathic effect in HuH-7 cells with the formation of round plaques. Studies of various recombinant viruses between JHM and A59 strains suggested that the ability of JHM to infect HuH-7 cells was determined by multiple viral genetic elements. Blocking the viral spike (S) protein with a neutralizing antibody or a soluble form of the MHV receptor inhibited infection of HuH-7 cells, suggesting that infection is mediated through the S protein. Transfection with the prototype mouse receptor, biliary glycoprotein, rendered HuH-7 cells susceptible to infection by other MHV strains as well, suggesting that JHM uses a receptor distinct from the classical MHV receptor to infect HuH-7 cells. Possible implications for human disease are discussed.

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.

Antibody prevents virus reactivation within the central nervous system.

The neurotropic JHM strain of mouse hepatitis virus (JHMV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. The immune response cannot completely eliminate virus, resulting in persistence associated with chronic ongoing CNS demyelination. The contribution of humoral immunity to viral clearance and persistent infection was investigated in mice homozygous for disruption of the Ig mu gene (IgM-/-). Acute disease developed with equal kinetics and severity in IgM-/- and syngeneic C57BL/6 (wt) mice. However, clinical disease progressed in IgM-/- mice, while wt mice recovered. Viral clearance during acute infection was similar in both groups, supporting a primary role of cell-mediated immunity in viral clearance. In contrast to wt mice, in which infectious virus was reduced to below detection following acute infection, increasing infectious virus was recovered from the CNS of the IgM-/- mice following initial clearance. No evidence was obtained for selection of variant viruses nor was there an apparent loss of cell-mediated immunity in the absence of Ab. Passive transfer of anti-JHMV Ab following initial clearance prevented reactivation of infectious virus within the CNS of IgM-/- mice. These data demonstrate the clearance of infectious virus during acute disease by cell-mediated immunity. However, immunologic control is not maintained in the absence of anti-viral Ab, resulting in recrudescence of infectious virus. These data suggest that humoral immunity plays no role in controlling virus during acute infection, but plays an important role in establishing and maintaining CNS viral persistence.

Mechanisms of viral clearance in perforin-deficient mice.

The roles of CD4+ T cells, IFN-gamma and TNF-alpha in viral clearance from the central nervous system (CNS) were examined in perforin gene deficient (PKO) mice. Depletion of CD4+ T cells from the PKO mice resulted in a significant 1 log10 PFU/gm increase in viral titer over control-treated PKO mice. PKO mice treated with anti-IFN-gamma mAb also had a significant 1 log10 increase in infectious virus whereas inhibition of TNF-alpha did not alter viral clearance or clinical disease in the PKO mice. These data suggest, in addition to perforin-mediated cytolysis, CD4+ T cells and IFN-gamma, but not TNF-alpha could contribute to JHMV clearance from the CNS.

Using a defective-interfering RNA system to express the HE protein of mouse hepatitis virus for studying viral pathogenesis.

We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin' esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express, HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.

The role of IL-10 in mouse hepatitis virus-induced demyelinating encephalomyelitis.

Interleukin 10 (IL-10) is an important anti-inflammatory cytokine. To examine its role in virus-induced encephalomyelitis, IL-10-deficient (IL-10 -/-) mice were infected with a neurotropic strain of mouse hepatitis virus (JHMV). JHMV-infected IL-10 -/- mice, compared to IL-4 -/- and syngeneic C57BL/6 mice, exhibited increased morbidity and mortality. Virus was cleared from the CNS of all groups of mice with equal kinetics by day 9 postinfection and the lack of either IL-4 or IL-10 did not alter the distribution of viral antigen, suggesting a lack of correlation between viral replication and the increased clinical disease in IL-10 -/- mice. In moribund IL-10 -/- mice, a moderate increase in mononuclear cell infiltration was correlated with increased expression of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase mRNAs. In the small percentage of IL-10 -/- mice that survived, no differences in either demyelination or inflammation were observed. Together, these results suggest that IL-10 is not required for viral clearance, and although it appears to be one of the mechanisms responsible for inhibiting the extent of inflammation in the CNS during acute JHMV infection, it has little role in the eventual resolution of CNS inflammatory responses.

Expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering RNA alters viral pathogenesis.

A defective-interfering (DI) RNA of mouse hepatitis virus (MHV) was developed as a vector for expressing MHV hemagglutinin/esterase (HE) protein. The virus containing an expressed HE protein (A59-DE-HE) was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. A similar virus (A59-DE-CAT) expressing the chloramphenicol acetyltransferase (CAT) was used as a control. These viruses were inoculated intracerebrally into mice, and the role of the HE protein in viral pathogenesis was evaluated. Results showed that all mice infected with parental A59 or A59-DE-CAT succumbed to infection by 9 days postinfection (p.i.), demonstrating that inclusion of the DI did not by itself alter pathogenesis. In contrast, 60% of mice infected with A59-DE-HE survived infection. HE- or CAT-specific subgenomic mRNAs were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the DI vector were expressed only in the early stage of viral infection. No significant difference in virus titer or viral antigen expression in brains was observed between A59-DE-HE- and A59-DE-CAT-infected mice, suggesting that virus replication in brain was not affected by the expression of HE. However, at day 3 p.i. there was a slight increase in the extent of inflammatory cell infiltration in the brains of the A59-DE-HE-infected mice. Surprisingly, virus titers in the livers of A59-DE-HE-infected mice were 3 log10 lower than that of the A59-DE-CAT-infected mice at day 6 p.i. Also, substantially less necrosis and viral antigen were detected in the livers of the A59-DE-HE-infected mice. This may account for the reduced mortality of these mice. The possible contribution of the host immune system to this difference in pathogenesis was analyzed by comparing the expression of four cytokines. Results showed that both tumor necrosis factor-alpha and interleukin-6 mRNAs increased in the brains of the A59-DE-HE-infected mice at day 2 p.i., whereas interferon-gamma and interleukin-1 alpha mRNAs were similar between A59-DE-HE- and A59-DE-CAT-infected mice. These data suggest that the transient expression of HE protein enhances an early innate immune response, possibly contributing to the eventual clearance of virus from the liver. This study indicates the feasibility of the DI expression system for studying roles of viral proteins during MHV infection.

Expression of interferon-gamma by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity.

A defective-interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV) was developed as a vector for expressing interferon-gamma (IFN-gamma). The murine IFN-gamma gene was cloned into the DI vector under the control of an MHV transcriptional promoter and transfected into MHV-infected cells. IFN-gamma was secreted into culture medium as early as 6 hr posttransfection and reached a peak level (up to 180 U/ml) at 12 hr posttransfection. The DI-expressed IFN-gamma (DE-IFN-gamma) exhibited an antiviral activity comparable to that of recombinant IFN-gamma and was blocked by a neutralizing monoclonal antibody against IFN-gamma. Treatment of macrophages with DE-IFN-gamma selectively induced the expression of the cellular inducible nitric oxide synthase and the IFN-gamma-inducing factor (IGIF) but did not affect the amounts of the MHV receptor mRNA. Antiviral activity was detected only when cells were pretreated with IFN-gamma for 24 hr prior to infection; no inhibition of virus replication was detected when cells were treated with IFN-gamma during or after infection. Furthermore, addition of IFN-gamma together with MHV did not prevent infection, but appeared to prevent subsequent viral spread. MHV variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to IFN-gamma treatment in vitro, with the most virulent strain being most resistant to IFN-gamma treatment. Infection of susceptible mice with DE-IFN-gamma-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the CNS and less virus replication, than that caused by virus containing a control DI vector. This study thus demonstrates the feasibility and usefulness of this MHV DI vector for expressing cytokines and may provide a model for studying the role of cytokines in MHV pathogenesis.

Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.

Perforin-deficient [perforin (-/-)] mice were infected with two strains of JHM virus (JHMV) to analyze the role of perforin-mediated cytotoxicity in acute lethal and subacute central nervous system (CNS) infections. During both acute and subacute infections, the overall mortality of the perforin (-/-) mice was not different from that of the controls. Perforin (-/-) mice survived longer than the controls, consistent with reduced morbidity. Both strains of virus were cleared from the perforin (-/-) mice as in the controls; however, the rate of clearance was delayed in the perforin (-/-) mice, indicating that perforin-mediated cytolysis is involved in viral clearance. The absence of perforin-mediated cytolysis did not prevent encephalomyelitis or extensive demyelination. Cells undergoing apoptosis were detected in the CNS of both the perforin (-/-) and control groups, indicating that perforin is not essential for programmed cell death. Neutralizing antibodies were not detected in either group of mice until day 9 postinfection, when the majority of the virus had been cleared. These data further confirm the importance of cell-mediated cytotoxicity and suggest that additional components of the immune response contribute to the clearance of JHMV from the CNS.

In vivo effects of T helper cell type 2 cytokines on macrophage antigen-presenting cell induction of T helper subsets.

SJL mice provide an interesting paradigm to examine the role(s) of APC in the differential induction of Th1 and Th2 cells. Immunization of young male SJL mice results in the preferential induction of Th2 cells, whereas Th1 cells are induced in age-matched female or older male SJL mice. The absence of Th1 responses in young male mice is associated with in vivo IL-4 and IL-10 down-regulating Mac-3+ APC priming of Th1 cells. The present report examines the mechanism of this APC-dependent induction of Th subsets. Examination of the surface expression of MHC class II, adhesion molecules (CD11a, CD11b, CD48, CD54, and CD102) or costimulatory molecules (CD24, CD80, and CD86) showed no differences between male- and female-derived Mac-3+ APC populations. In addition, no differences were detected in IL-1alpha, IL-1beta, IL-18, TNF-alpha, or IL-12 p35 mRNA expression. However, reduced expression of both IL-10 and IL-12 p40 mRNA were found in Mac-3+ cells from male mice compared with those in Mac-3+ cells from female mice. Anti-IL-4 or anti-IL-10 mAb treatment of young male donor mice eliminated the reduction of both IL-10 and IL-12 p40 mRNA, suggesting that the Th2 inducer phenotype is related to a decreased IL-12 secretion. Consistent with this idea, fewer IL-12 p40-secreting Mac-3+ cells were found in male mice compared with female mice, and treatment with rIL-12 resulted in the priming of Th1 cells in male mice. These data suggest that increased Th2 cytokines in vivo before encounter with Ag inhibit APC expression of IL-12, resulting in the preferential induction of Th2 cells in male SJL mice.