Figure 1.
Kinetics of EBV DNA and expression patterns of EBV-related genes in human tonsil tissue explants infected with B95–8.
Culture medium was changed every 3 days, and collected medium was centrifuged. Cell-free supernatants and cells collected from culture medium were used for quantification of EBV DNA by real-time PCR assay and quantification of viral mRNA by real-time RT-PCR assay. Data are presented as mean ± standard error of the mean. A) Tissue blocks on top of collagen sponge gels in a six-well plate. B) Kinetics of EBV DNA in cell-free supernatants. Average data were obtained from tissues derived from 12 donors. C) Plots of accumulated EBV DNA in cell-free culture medium after day 12 post-infection (n = 12). D) Kinetics of EBV DNA in cells from medium (n = 12). E) Levels of EBV-related gene expressions in cells at 3, 12, and 24 days post-infection (n = 4).
Figure 2.
Kinetics of EBV DNA and expression patterns of EBV-related genes in human tonsil tissue explants infected with EGFP-EBV.
Cell-free supernatants and cells collected from culture medium were used for quantification of EBV DNA by real-time PCR assay and quantification of viral mRNA by real-time RT-PCR assay. Data are presented as mean ± standard error of the mean. A) Plots of accumulated EBV DNA in cell-free culture medium after 12 days post-infection (n = 8). B) Plots of accumulated EBV DNA in cells from medium (n = 2). C) Levels of EBV-related genes expressions in cells at 3 and 24 days post-infection (n = 3).
Figure 3.
Histological analyses of tonsil tissues infected with B95–8 using immunostaining and in situ hybridization with EBER-RNA.
Samples were obtained from EBV-infected tissues at 24 days post-infection. Pictures are representative of results from four experiments. A) Low-power view of tonsillar lymphoid tissue (HE stain). Magnification, ×100. B) Low-power view of CD3+ lymphocytes in an interfollicular area (CD3 stain). Magnification, ×100. C) Low-power view of CD20+ lymphocytes in a follicular area (CD20 stain). Magnification, ×100. D) High-power view of anti-follicular dendritic cell (FDC)+ in a follicular area (anti-FDC stain). Magnification, ×400. E) Low-power view of EBER+ lymphocytes (EBER ISH stain). Magnification, ×100. F) High-power view of EBER+ lymphocytes in an interfollicular area (EBER ISH stain). Magnification, ×400. G) High-power view of BZLF1+ lymphocytes (arrows) in an interfollicular area (BZLF1 stain). Magnification, ×400.
Figure 4.
Phenotype of EGFP+ cells in human tonsil tissue infected with EGFP-EBV.
At 15–24 days post-infection, single cell suspensions were dissected from tissue blocks. Cells were stained with phycoerythrin (PE)-labeled anti-CD19, CD3, CD56 monoclonal antibodies, then analyzed by flow cytometry. A) A representative experiment is shown (n = 4). Density plots represent CD19, CD3, CD56 versus EGFP for control and EGFP-EBV-infected tissues at 24 days post-infection. Numbers in quadrants indicate percentages of lymphocytes for each surface immunophenotype. B) Mean proportion of EGFP+ CD19+ cells among CD19+ cells at 15–24 days post-infection. Average data were obtained from 27 blocks of tissue derived from four donors. Results represent mean ± standard error of the mean. ctrl, control (uninfected tonsil tissues).
Figure 5.
Phenotype of EGFP+ cells in human tonsil tissue infected with EGFP-EBV using flow cytometry.
Samples were obtained from EBV-infected tissues at 24 days post-infection. EGFP+ (red) and EGFP− (grey) MNCs were gated and plotted on quadrants as PE-labeled and PC5-labeled surface antigens. A representative experiment is shown (n = 2). Numbers in quadrants indicate percentages of EGFP+ lymphocytes for each surface immunophenotype.
Figure 6.
Inhibition by acyclovir (ACV) of EBV replication in human tonsillar tissues explants.
Human tonsillar tissues were infected with B95–8 and treated with ACV at various concentrations. Antiviral activity of ACV was evaluated by comparing viral replication in ACV-treated tissues with that in untreated donor-matched control tissues. Data are presented as means ± standard errors of mean. A) Kinetics of EBV replication were measured by real-time PCR assay for viral DNA in culture medium (n = 3). B) EBV DNA was quantified in cells at 24 days post-infection (n = 3). C) Comparison of the number of EBV-infected cells in the presence or absence of ACV at 24 days post-infection using FISH assay (n = 1).