Simulating Epstein-Barr virus infection with C-ImmSim.

MOTIVATION: Epstein-Barr virus (EBV) infects greater than 90% of humans benignly for life but can be associated with tumors. It is a uniquely human pathogen that is amenable to quantitative analysis; however, there is no applicable animal model. Computer models may provide a virtual environment to perform experiments not possible in human volunteers. RESULTS: We report the application of a relatively simple stochastic cellular automaton (C-ImmSim) to the modeling of EBV infection. Infected B-cell dynamics in the acute and chronic phases of infection correspond well to clinical data including the establishment of a long term persistent infection (up to 10 years) that is absolutely dependent on access of latently infected B cells to the peripheral pool where they are not subject to immunosurveillance. In the absence of this compartment the infection is cleared. AVAILABILITY: The latest version 6 of C-ImmSim is available under the GNU General Public License and is downloadable from www.iac.cnr.it/~filippo/cimmsim.html

EBV and systemic lupus erythematosus: a new perspective.

We have proposed that EBV uses mature B cell biology to access memory B cells as a site of persistent infection. A central feature of this model is that EBV adapts its gene expression profile to the state of the B cell it resides in and that the level of infection is stable over time. This led us to question whether changes in the behavior or regulation of mature B cells would alter the state of EBV persistence. To investigate this, we studied the impact of systemic lupus erythematosus (SLE), a disease characterized by immune dysfunction, on EBV infection. We show that patients with SLE have abnormally high frequencies of EBV-infected cells in their blood, and this is associated with the occurrence of SLE disease flares. Although patients with SLE have frequencies of infected cells comparable to those seen in immunosuppressed patients, in SLE the effect was independent of immunosuppressive therapy. Aberrant expression of viral lytic (BZLF1) and latency (latency membrane proteins 1 and 2a) genes was also detected in the blood of SLE patients. We conclude that the abnormal regulation of EBV infection in SLE patients reflects the sensitivity of the virus to perturbation of the immune system.

Quantitative detection of viral gene expression in populations of Epstein-Barr virus-infected cells in vivo.

The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this technique involves limiting dilution analysis, it is possible to define which viral transcription programs are being used at the single-cell level. This assay takes 3-4 d to complete and involves the following steps: (1) sample preparation and isolation of the cell population of interest; (2) DNA-PCR limiting dilution analysis to determine the frequency of infected cells within the cell population; (3) RNA isolation; (4) cDNA synthesis; (5) PCR; (6) visualization of PCR products by Southern blotting; and (7) calculations. As an example, we have used PBMCs from the blood of an acute infectious mononucleosis patient. However, this technique can be applied to other cell populations, such as B cells, and other patient groups, such as healthy long-term virus carriers and immunosuppressed organ transplant recipients.

Acute infection with Epstein-Barr virus targets and overwhelms the peripheral memory B-cell compartment with resting, latently infected cells.

In this paper we demonstrate that during acute infection with Epstein-Barr virus (EBV), the peripheral blood fills up with latently infected, resting memory B cells to the point where up to 50% of all the memory cells may carry EBV. Despite this massive invasion of the memory compartment, the virus remains tightly restricted to memory cells, such that, in one donor, fewer than 1 in 10(4) infected cells were found in the naive compartment. We conclude that, even during acute infection, EBV persistence is tightly regulated. This result confirms the prediction that during the early phase of infection, before cellular immunity is effective, there is nothing to prevent amplification of the viral cycle of infection, differentiation, and reactivation, causing the peripheral memory compartment to fill up with latently infected cells. Subsequently, there is a rapid decline in infected cells for the first few weeks that approximates the decay in the cytotoxic-T-cell responses to viral replicative antigens. This phase is followed by a slower decline that, even by 1 year, had not reached a steady state. Therefore, EBV may approach but never reach a stable equilibrium.

Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.

Epstein-Barr virus (EBV) is a herpesvirus that establishes a lifelong, persistent infection. It was first discovered in the tumor Burkitt's lymphoma (BL). Despite intensive study, the role of EBV in BL remains enigmatic. One striking feature of the tumor is the unique pattern of viral latent protein expression, which is restricted to EBV-encoded nuclear antigen (EBNA) 1. EBNA1 is required to maintain the viral genome but is not recognized by cytotoxic T cells. Consequently, it was proposed that this expression pattern was used by latently infected B cells in vivo. This would be the site of long-term, persistent infection by the virus and, by implication, the progenitor of BL. We now know that EBV persists in memory B cells in the peripheral blood and that BL is a tumor of memory cells. However, a normal B cell expressing EBNA1 alone has been elusive. Here we show that most infected cells in the blood express no detectable latent mRNA or proteins. The exception is that when infected cells divide they express EBNA1 only. This is the first detection of the BL viral phenotype in a normal, infected B cell in vivo. It suggests that BL may be a tumor of a latently infected memory B cell that is stuck proliferating because it is a tumor and, therefore, constitutively expressing only EBNA1.

The dispersal of mucosal memory B cells: evidence from persistent EBV infection.

We have used latent infection with the human herpesvirus Epstein-Barr virus to track the dispersal of memory B cells from the mucosal lymphoid tissue of Waldeyer's ring (tonsils/adenoids). EBV is evenly distributed between the memory compartments of Waldeyer's ring and the peripheral blood. However, it has an approximately 20-fold higher preference for Waldeyer's ring over the spleen or mesenteric lymph nodes. These observations are consistent with a model whereby the virus preferentially establishes persistent infection within memory B cells from Waldeyer's ring. The virus then colonizes the entire peripheral lymphoid system, at a low level, by trafficking with these memory B cells as they circulate through the body and back to Waldeyer's ring. This pathway may reflect that of normal memory B cells derived from nasopharyngeal and other mucosal lymph nodes.