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. 2004 May;78(10):5194-204.
doi: 10.1128/jvi.78.10.5194-5204.2004.

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

Donna Hochberg  1 , Tatyana Souza, Michelle Catalina, John L Sullivan, Katherine Luzuriaga, David A Thorley-Lawson
Affiliations

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

Donna Hochberg et al. J Virol. 2004 May.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Schematic drawing of a model for EBV persistence. Virions enter the mucosal surface of the nasopharynx through saliva. The virus enters the lymphoid tissue of Waldeyer's ring, where it infects resting naive B cells and drives them to become proliferating lymphoblasts through expression of nine latent proteins (the growth program) (46). These blasts can then differentiate into resting memory cells by a process analogous to the germinal center reaction by using the default program (46). Once in resting memory cells, all viral protein expression ceases (the latency program) (16, 46). Latently infected memory cells circulate in the periphery and return to the lymphoid tissue, where at some unknown rate, they differentiate into plasma cells and release infectious virus to initiate a new round of infection (Laichalk and Thorley-Lawson, submitted). Each stage of the cycle is subject to immunosurveillance (CTL against infected cells and antibody against virions), with the exception of the memory cells, which are invisible because they express no viral proteins (16). In the absence of an immune response, the cycle will amplify until the memory B compartment is filled with latently infected cells. Once the immune response is activated, the cycle is dramatically reduced or perhaps even completely blocked and the reservoir of latently infected memory cells decays.
FIG. 2.
FIG. 2.
EBV-infected cells in the periphery of AIM patients are CD27 positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
FIG. 3.
FIG. 3.
EBV-infected cells in the periphery of AIM patients are IgD negative. PBMCs were stained for expression of CD20, a pan-B-cell marker, and IgD (upper panel). The naive (IgD positive) and memory (IgD negative) B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
FIG. 4.
FIG. 4.
Cell cycle stage of EBV-infected cells in the blood of AIM patients. B cells from an EBV-transformed cell line (A) or from the blood of AIM patients (B) were stained with the vital DNA dye Hoechst 33342. The AIM B cells were then separated into the G0/G1 and S/G2/M fractions by FACS, and the frequency of infected cells in each population was measured by limiting dilution DNA PCR (C).
FIG. 5.
FIG. 5.
B cells, replicating EBV, are rare in the blood of AIM patients. PBMCs (A) or purified B cells (B) were isolated, and the frequency of virus-infected cells expressing the BZLF1 or EBER1 genes was measured by performing a limiting dilution RT-PCR assay. Serial dilutions of cells were prepared, and multiple samples of each dilution were tested for expression of each RNA by RT-PCR. BZLF1 is the gene that initiates viral replication, and EBER1 is believed to be widely expressed in EBV-infected cells. The number of infected cells tested was predetermined by limiting dilution DNA PCR. (C) A control experiment with an EBV-positive cell line demonstrating that the technique can detect a single infected cell expressing BZLF1.
FIG. 6.
FIG. 6.
Comparison of frequencies of EBV-infected B cells in blood of AIM patients, immunosuppressed allograft patients, and healthy carriers. The AIM data (n = 20) (open bars) are from the patients listed in Table 4, the data from the immunosuppressed allograft patients (n = 25) (filled bars) have been published previously (2). The healthy carriers (n = 46) (hatched bar) are a new cohort, but similar data have been published previously by our laboratory (22, 29, 33) and are not detailed here. The frequencies of infected cells in this figure are expressed as the natural logs of the number of infected cells in 107 total B cells, not memory B cells.
FIG. 7.
FIG. 7.
Detection of high levels of EBV-infected memory B cells in blood of AIM patients. IgD-negative memory B cells were isolated as described in the legend to Fig. 3. Serial dilutions of the cells were then prepared (e.g., 0.75 means that, on average, 7.5 of 10 replicates will have 1 cell), and multiple replicates of each dilution were tested for the presence of EBV either by DNA PCR and Southern blotting for the viral DNA (A) (patient 1 in Table 4) or by real-time PCR for EBER1 RNA (B) (patient 2 in Table 4).
FIG. 8.
FIG. 8.
Time course of resolution of EBV infection in blood of AIM patients. The frequencies of infected cells are expressed as the natural logs of the number of infected cells per 107 memory B cells. The dashed horizontal line represents the upper limit, and unbroken line represents the mean of the number of infected memory B cells detected in healthy carriers.
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