Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs. RESULTS: Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20-22 h p.i.), but the logarithmic infection phase (days 2-3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-gamma (IFN-gamma), as well as the more-potent experimental antiviral agent AK-2. CONCLUSION: The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.

Polyclonal hypergammaglobulinemia and formation of hydrophobic immune complexes in porcine reproductive and respiratory syndrome virus-infected and uninfected pigs.

Infection of young conventional, domestic pigs with porcine reproductive and respiratory syndrome virus (PRRSV) strains VR2332 and JA142 resulted in a rapid, progressive increase in serum IgG reaching maximum levels of 20-30 mg/mL at about 3 weeks post infection (p.i.), which were maintained until at least 63 days p.i., whereas the level of serum IgG remained at 4-6 mg/mL in sham infected pigs. In most of the VR2332 and JA142-infected pigs hypergammaglobulimenia was associated with the formation of hydrophobic, 150-300-kDa IgG-containing immune complexes that bound in the presence of 0.1% Tween 20 to ELISA plates that were not coated with any antigen. The ELISA plate-binding activity remained low in most infected pigs, but reached high levels in some JA142-infected pigs. Binding of the immune complexes was also observed, but at a lower level, to uncoated ELISA plates in the peptide ELISA for anti-PRRSV Abs. The immune complexes bound to uncoated ELISA plates with a much lower affinity than Abs to plates coated with peptides containing the appropriate epitopes. The immune complexes also bound to HerdChek ELISA plates, but because of low binding affinity for these plates, the bound complexes were removed by the repeated washes with Tween 20 solution. Overall the PRRSV-induced hypergammaglobulinemia and generation of ELISA plate-binding immune complexes resembled those observed in mice infected with the closely related lactate dehydrogenase-elevating virus (LDV) and thus, like the latter, seem a result of a polyclonal activation of B cells. We also found that sera of a group of older sows possessed high levels of IgG as well as of ELISA plate-binding immune complexes, in spite of being PRRSV infection negative by all criteria presently available. On the other hand, sera from wild hogs contained no ELISA plate-binding IgG in spite of possessing high total serum IgG levels.

Lactate dehydrogenase-elevating virus induces apoptosis in cultured macrophages and in spinal cords of C58 mice coincident with onset of murine amyotrophic lateral sclerosis.

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.

Glucocorticoid regulation of lactate dehydrogenase-elevating virus replication in macrophages.

Lactate dehydrogenase-elevating virus (LDV) is a macrophage-tropic arterivirus which generally causes a persistent viremic infection in mice. LDV replication in vivo seems to be primarily regulated by the extent and dynamics of a virus-permissive macrophage population. Previous studies have shown that glucocorticoid treatment of chronically LDV-infected mice transiently increases viremia 10-100-fold, apparently by increasing the productive infection of macrophages. We have further investigated this phenomenon by comparing the effect of dexamethasone on the in vivo and in vitro replication of two LDV quasispecies that differ in sensitivity to immune control by the host. The single neutralizing epitope of LDV-P is flanked by two N-glycans that impair its immunogenicity and render LDV-P resistant to antibody neutralization. In contrast, replication of the neuropathogenic mutant LDV-C is suppressed by antibody neutralization because its epitope lacks the two protective N-glycans. Dexamethasone treatment of mice 16 h prior to LDV-P infection, or of chronically LDV-P infected mice, stimulated viremia 10-100-fold, which correlated with an increase of LDV permissive macrophages in the peritoneum and increased LDV infected cells in the spleen, respectively. The increase in viremia occurred in the absence of changes in total anti-LDV and neutralizing antibodies. The results indicate that increased viremia was due to increased availability of LDV permissive macrophages, and that during a chronic LDV-P infection virus replication is strictly limited by the rate of regeneration of permissive macrophages. In contrast, dexamethasone treatment had no significant effect on the level of viremia in chronically LDV-C infected mice, consistent with the view that LDV-C replication is primarily restricted by antibody neutralization and not by a lack of permissive macrophages. beta-Glucan, the receptor of which is induced on macrophages by dexamethasone treatment, had no effect on the LDV permissiveness of macrophages.

Hydrophobic IgG-containing immune complexes in the plasma of autoimmune MRL/lpr mice, lactate dehydrogenase-elevating virus-infected mice, and pigs: association with transforming growth factor-beta and pH-dependent amplification.

Persistent infection of mice with lactate dehydrogenase-elevating virus (LDV) is associated with polyclonal B cell activation, autoimmunity, and circulating hydrophobic IgG-containing immune complexes (ICs), which bind to the surfaces of uncoated ELISA plates in the presence of 0.05% Tween 20. We demonstrate here that hydrophobic IgG-containing ICs also appear naturally in the plasma of autoimmune MRL/lpr mice. These and the similar hydrophobic ICs of LDV-infected mice as well as pigs coincide on ELISA plate surfaces with TGF-beta, apparently in the form of an IgG-TGF-beta complex. Circulating hydrophobic IgG-containing ICs are also susceptible to considerable amplification in vitro by exposure to alkaline conditions. By this latter method, the fraction of in vivo hydrophobic IgG, relative to the maximum in vitro chemically inducible IgG, was found to be about 20% in the plasma of LDV-infected mice, 5% in normal mouse plasma, and less than about 2% in pig plasma. These results indicate the potential for both chemically induced and protein-binding contributions to the generation of hydrophobic IgG-containing molecules, and have implications for immunopathological mechanisms in autoimmunity and persistent virus infections.

Current concepts in multiple sclerosis: Part II.

Multiple sclerosis (MS) is a complex and challenging autoimmune disease of the central nervous system, affecting approximately 0.1% of the US population. Evidence to date suggests that viral infection triggers autoimmune attack against nerve cells in genetically-susceptible individuals. Neurologic deficits then appear, typically with a variable course and episodes of remission. Partial treatment success has been obtained with immunomodulating agents, such as interferon-beta and intravenous immunoglobulins. Current research is directed at elucidating potential viral causes of MS, as well as the interaction of host genes with the immunopathogenic mechanisms involved in MS. In the future, it may be possible to vaccinate susceptible individuals against MS, as well as refine immunomodulation therapy for the treatment of MS.

Current concepts in multiple sclerosis: Part I.

Multiple sclerosis (MS) is a complex and challenging autoimmune disease of the central nervous system, affecting approximately 0.1% of the US population. Evidence to date suggests that viral infection triggers autoimmune attack against nerve cells in genetically-susceptible individuals. Neurologic deficits then appear, typically with a variable course and episodes of remission. Partial treatment success has been obtained with immunomodulating agents, such as interferon-beta and intravenous immunoglobulins. Current research is directed at elucidating potential viral causes of MS, as well as the interaction of host genes with the immunopathogenic mechanisms involved in MS. In the future, it may be possible to vaccinate susceptible individuals against MS, as well as refine immunomodulation therapy for the treatment of MS.

N-glycans on the short ectodomain of the primary envelope glycoprotein play a major role in the polyclonal activation of B cells by lactate dehydrogenase-elevating virus.

The common biologically cloned isolates of lactate dehydrogenase-elevating virus (LDV-P and LDV-vx) invariably cause a polyclonal activation of B cells in immunocompetent mice. It is recognized by an at least 10-fold increase in plasma IgG2a levels and the de novo formation of immune complexes that most likely consist of autoantibodies and their antigens. The present study indicates that three closely spaced N-glycans on the short ectodomain of the primary envelope glycoprotein, VP-3P, of LDV-P/vx, play a major role in inducing the polyclonal proliferation of B cells. IFN-gamma then seems to mediate the differentiation of the activated B cells to IgG2a-producing plasma cells. These conclusions are based on the finding that the IgG2a hypergammaglobulinaemia and immune complex formation were much lower in mice that were infected with LDV variants (LDV-C and LDV-v) whose VP-3P ectodomains lack two of the three N-glycans than in LDV-P/vx infected mice. In contrast, the VP-3P ectodomains of three neutralization escape variants of LDV-C/v whose VP-3P ectodomains possess three N-glycosylation sites caused a polyclonal activation of B cells comparable to that of LDV-P/vx.