Patricia E. Tam

Multiple Viral Determinants Mediate Myopathogenicity in Coxsackievirus B1-Induced Chronic Inflammatory Myopathy

ABSTRACT Mice infected with myopathic coxsackievirus B1 Tucson (CVB1T) develop chronic inflammatory myopathy (CIM) consisting of hind limb weakness and inflammation. Amyopathic virus variants are infectious but attenuated for CIM. In this report, viral clones, chimeras, and sequencing were used to identify viral determinants of CIM. Chimeras identified several regions involved in CIM and localized a weakness determinant to nucleotides 2493 to 3200 of VP1. Sequencing of multiple clones and viruses identified five candidate determinants that were strictly conserved in myopathic viruses with one located in the 5′ untranslated region (UTR), three in the VP1 capsid, and one in the 3C protease. Taken together, these studies implicate Tyr-87 and/or Val-136 as candidate determinants of weakness. They also indicate that there are at least two determinants of inflammation and one additional determinant of weakness encoded by myopathic CVB1T.

Coxsackievirus B1-induced chronic inflammatory myopathy: differences in induction of autoantibodies to muscle and nuclear antigens by cloned myopathic and amyopathic viruses.

Infection of susceptible strains of mice with the myopathic Tucson strain of coxsackievirus B1 (CVB1(T)) leads to the development of chronic inflammatory myopathy (CIM). The underlying mechanism of CIM appears to be immunopathic, but it is not known whether autoimmunity is involved. The objectives of this study were to determine whether autoantibodies are produced and whether they correlate with the pathology of CIM. Mice were infected with either a myopathic (MP1.23 or MP1.24) or an amyopathic (AMP2.17) CVB1(T) cloned virus. The two myopathic (MP) viruses cause CIM, whereas the amyopathic (AMP) virus, derived from a variant of the same parent, causes the same acute disease but does not cause CIM. Antimuscle IgG was found in 51% of MP1.23-infected and 58% of MP1.24-infected mice but in just 18% of mice infected with AMP2.17 and in 10% of controls (MP vs AMP: chi(2), P < or =.006). Several staining patterns were observed, indicating that autoantibodies of multiple specificities were produced. Antinuclear antibodies were found in 57% of MP1.23-infected and 27% of MP1.24-infected mice but were rare in mice infected with AMP2.17 (0%) or in controls (4%) (MP vs AMP: chi(2), P < or =.01). Antiviral-antibody titers were higher with MP virus than with AMP virus (ANOVA, P <.001). A trend toward an association between antiviral antibody or autoantibodies and the presence or severity of clinical measures of CIM was noted but was not significant. These data suggest that the autoantibodies do not mediate muscle disease but are an independent manifestation of an immunopathic response induced by infection with MP but not AMP CVB1(T).

Genetic determinants of susceptibility to coxsackievirus B1-induced chronic inflammatory myopathy: effects of host background and major histocompatibility complex genes.

Infection of outbred CD-1 mice with the Tucson strain of coxsackievirus B1 (CVB1T) leads to the development of chronic hind limb weakness and associated inflammatory muscle disease. Host factors that influence susceptibility have not been studied in this mouse model of chronic inflammatory myopathy (IM). Therefore, the pathogenesis was examined by using different inbred strains of mice. Initially, seven strains of mice with either the H-2d or H-2b major histocompatibility complex (MHC) haplotype were evaluated. All strains showed similar levels of acute mortality caused by viral infection, but chronic weakness or inflammation did not develop in two strains with the B6 background, regardless of their MHC haplotype. In susceptible mice, weakness was more likely to develop in the H-2d strains than in mice with the H-2b haplotype. Based on these results, H-2 congenic strains of the susceptible B10 background (C57BL/10 and B10.D2) and the resistant B6 background (C57BL/6 and B6.C-H2d) were examined in greater detail. During acute infection, the kinetics and degree of viral replication in hind limb muscle were similar among B6 and B10 strains. By 4 weeks after infection, more intense chronic muscle inflammation and pathology were observed in susceptible B10 mice of the H-2d haplotype than in those of the H-2b haplotype. Resistant B6 mice did not show signs of inflammation or calcification, but they did exhibit some myopathic features, including centralized nuclei and variations in myofiber size and shape. These changes were less common in resistant B6 mice than in B10 strains but were significant when compared with changes in uninfected controls. Viral RNA persistence and elevated titers of antiviral IgG were more prevalent in but not restricted to susceptible strains. These studies demonstrate that host background genes confer resistance to chronic IM but also that MHC genes influence disease severity. They also reveal that susceptibility to acute CVB1T infection is under different genetic control than that which mediates development of chronic post-viral IM.

Differential inhibition of mitogenic responsiveness by monoclonal antibodies to β2-microglobulin

A panel of 10 monoclonal antibodies (MoAbs) to human beta 2-microglobulin (beta 2m) was used to evaluate the modulation of lymphocyte activation induced by different mitogenic stimuli. All 10 MoAbs inhibited proliferative responses of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and allogeneic cells in mixed lymphocyte culture (MLC), although some MoAbs were inhibitory at much lower concentrations than others. No enhancement or direct mitogenicity was observed, but at low MoAb concentrations a delayed peak response sometimes occurred. Differentiation of B cells in PWM-stimulated PBMC cultures was also inhibited as measured by reduced accumulation of supernatant IgM and IgG. Anti-beta 2m MoAb did not interfere with the binding of PHA or PWM to PBMC, and membrane mobility as judged by subsequent capping of these lectins also appeared to be normal. Furthermore, anti-beta 2m was inhibitory when added 24 hr prior to peak responsiveness, and proliferative responses to the phorbol ester PMA in combination with ionomycin were also inhibited by MoAb, indicating that membrane-mediated events were not the target of inhibition. A comparison of the inhibitory effects of anti-beta 2m MoAb on activation by different stimuli revealed that PWM and MLC responses were much more sensitive to inhibition followed by, in order of decreasing inhibition, Con A, PHA, ionomycin alone, and PMA/ionomycin. A MoAb to a monomorphic determinant of HLA-A, B, C exhibited the same inhibitory trend, suggesting that the mechanism of inhibition was the same as for anti-beta 2m MoAbs. No inhibition was observed when PBMC were stimulated by PMA alone, suggesting that the MoAbs have little effect on activation mediated by protein kinase C but may preferentially affect the calcium-dependent pathway of activation. Thus, this differential inhibition observed with different stimuli may reflect the relative contribution of class I antigens to lymphocyte activation by a particular mitogen.