Chronic hepatitis C virus infection breaks tolerance and drives polyclonal expansion of autoreactive B cells.

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (V(H)) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the V(H) genes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.

Germinal center B cells latently infected with Epstein-Barr virus proliferate extensively but do not increase in number.

In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo.

Epstein-Barr virus infection is not a characteristic feature of multiple sclerosis brain.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) that is thought to be caused by a combination of genetic and environmental factors. To date, considerable evidence has associated Epstein-Barr virus (EBV) infection with disease development. However, it remains controversial whether EBV infects multiple sclerosis brain and contributes directly to CNS immunopathology. To assess whether EBV infection is a characteristic feature of multiple sclerosis brain, a large cohort of multiple sclerosis specimens containing white matter lesions (nine adult and three paediatric cases) with a heterogeneous B cell infiltrate and a second cohort of multiple sclerosis specimens (12 cases) that included B cell infiltration within the meninges and parenchymal B cell aggregates, were examined for EBV infection using multiple methodologies including in situ hybridization, immunohistochemistry and two independent real-time polymerase chain reaction (PCR) methodologies that detect genomic EBV or the abundant EBV encoded RNA (EBER) 1, respectively. We report that EBV could not be detected in any of the multiple sclerosis specimens containing white matter lesions by any of the methods employed, yet EBV was readily detectable in multiple Epstein-Barr virus-positive control tissues including several CNS lymphomas. Furthermore, EBV was not detected in our second cohort of multiple sclerosis specimens by in situ hybridization. However, our real-time PCR methodologies, which were capable of detecting very few EBV infected cells, detected EBV at low levels in only 2 of the 12 multiple sclerosis meningeal specimens examined. Our finding that CNS EBV infection was rare in multiple sclerosis brain indicates that EBV infection is unlikely to contribute directly to multiple sclerosis brain pathology in the vast majority of cases.

The intersection of Epstein-Barr virus with the germinal center.

The current model of Epstein-Barr virus (EBV) infection and persistence in vivo proposes that EBV uses the germinal center (the GC model) to establish a quiescent latent infection in otherwise-normal memory B cells. However, the evidence linking EBV-infected cells and the GC is only indirect and limited. Therefore, a key portion of the model, that EBV-infected cells physically reside and participate in GCs, has yet to be verified. Furthermore, recent experiments suggested that upon infection of GC cells the viral growth latency transcription program is dominant and GC functionality and phenotype are ablated, i.e., EBV infection is not consistent with GC function. In this study we show that in vivo, EBV-infected B cells in the tonsils retain expression of functional and phenotypic markers of GC cells, including bcl-6 and AID. Furthermore, these cells are physically located in the GC and express a restricted form of latency, the default latency program. Thus, the EBV default latency transcription program, unlike the growth latency program, is consistent with the retention of GC functionality in vivo. This work verifies key components of the GC model of EBV persistence and suggests that EBV and the GC can interact to produce the latently infected memory cells found in the periphery. Furthermore, it identifies latently infected GC B cells as a potential pathogenic nexus for the development of the EBV-positive, GC-associated lymphomas Hodgkin's disease and Burkitt's lymphoma.