Detection of Epstein-Barr virus genomes in peripheral blood B cells from solid-organ transplant recipients by fluorescence in situ hybridization

Abstract

Resolution of Epstein-Barr Virus (EBV) infection in pediatric solid-organ transplant recipients often leads to an asymptomatic carrier state characterized by a persistently elevated circulating EBV load that is 2 to 4 orders of magnitude greater than the load typical of healthy latently infected individuals. Elevated EBV loads in immunosuppressed individuals are associated with an increased risk for development of posttransplant lymphoproliferative disease. We have performed fluorescence in situ hybridization (FISH) studies with peripheral blood B cells from carriers of persistent EBV loads in order to directly quantitate the number of EBV genomes per infected cell. Patients were assigned to two groups on the basis of the level of the persistent load (low-load carriers, 8 to 200 genomes/10(5) peripheral blood lymphocytes; high-load carriers, >200 genomes/10(5) peripheral blood lymphocytes). FISH analysis revealed that the low-load carriers predominantly had circulating virus-infected cells harboring one or two genome copies/cell. High-load carriers also had cells harboring one or two genome copies/cell; in addition, however, they carried a distinct population of cells with high numbers of viral genome copies. The increased viral loads correlated with an increase in the frequency of cells containing high numbers of viral genomes. We conclude that low-load carriers possess EBV-infected cells that are in a state similar to normal latency, whereas high-load carriers possess two populations of virus-positive B cells, one of which carries an increased number of viral genomes per cell and is not typical of normal latency.

FIG. 1.

FISH assay for EBV with control cell lines. In situ hybridization with the WWYH DNA probe for the detection of the EBV genome was performed with BJAB cells (negative control); Namalwa cells (positive control), which have two integrated genomes per cell; and B95-8CR cells (positive control), which have multiple copies of episomal DNA. The percentages of cells with different numbers of hybridization spots (copies of the EBV genome) per cell for each cell line are summarized below each photo.

FIG. 2.

EBV genomes detected in peripheral blood B cells from chronic low-load carriers. Examples of cells with typical patterns of fluorescence were derived from patient 3 (D and G), patient 4 (A, B, E, and F), and patient 5 (C). All cells contained low numbers of genomes per cell. (H) Summary of the distribution of the number of genomes per cell for all persistent low-load carriers expressed as a percentage of all EBV-positive cells present.

FIG. 3.

EBV genomes detected in peripheral blood B cells from chronic high-load carriers. Examples of cells with typical patterns of fluorescence were derived from patient 6 (E and H), patient 7 (B and G), and patient 10 (A, C, D, and F), which revealed a mixed population of cells with 5 or fewer genomes per cell and cells with 20 to 30 genomes per cell. (I) Summary of the distribution of the number of genomes per cell for persistent high-load carriers as a percentage of all EBV-positive cells present.

FIG. 4.

EBV genomes detected in peripheral blood B cells from patients with a recent diagnosis of PTLD. Examples of cells with typical patterns of fluorescence were derived from patient 12 (A to E), patient 13 (F to H), patient 14 (J), and patient 15 (I), which revealed a predominance of cells with high numbers of genome copies (20 to 30 genomes per cell). (K) Summary of the distribution of the number of genomes per cell for all PTLD patients as a percentage of all EBV-positive cells present.

FIG. 5.

(A) Percentage of CD19⁺ cells that were EBV positive in low- and high-load carriers as well as PTLD patients. Open bars, cells with 10 or fewer genomes per cell; closed bars, cells with greater than 10 genomes per cell. (B) Relative distributions of cells with low and high viral genome copy numbers in low- and high-load carriers, showing the predominance of cells with high copy numbers in patients with loads greater than 200 copies/10⁵ PBLs. The ratios of the percentage of cells with high copy numbers to the percentage of cells with low copy numbers were 0.06, 0.7, and 1.2 for persistent low-load carriers, persistent high-load carriers, and PTLD patients, respectively.

FIG. 6.

Comparison of the distribution of the number of EBV episomes per cell predicted with a random assortment model and the observed distribution of EBV episomes per cell. (A) Expected distribution of the number of genomes per cell predicted with the random assortment model after cells become infected with a single virus particle and the viral episome replicates during cell proliferation. The plots represent the calculated distribution of the number of episomes per infected cell expected after B cells have proliferated for 2 (circles), 3 (squares), or 10 (triangles) generations. (B) Observed distribution of the number of EBV episomes per infected cell in the peripheral blood of low-load carriers (circles), high-load carriers (squares), and PTLD patients (triangles).