Rapid determination of Epstein–Barr virus latent or lytic infection in single human cells using in situ hybridization

Abstract

Epstein-Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein-Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein-Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.

Quantitative analysis of Epstein-Barr virus (EBV)-related gene expression in patients with chronic active EBV infection

“…These results are consistent with those from previous reports (Leenman et al, 2004;Tao et al, 1998;Tsuge et al, 1999;Zhang et al, 2003), indicating the reliability of our system. We evaluated the sensitivity for each latent EBV gene using a cell mixture containing 1610 6 EBV-negative BJAB cells and 10-fold serial dilutions of LCL-1 with latency III.…”

Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing

“…19, 20, 21 We estimated 80 EBV and 370 mitochondrial copies per DAUDI cell based on coverage analyses, which is in accordance with previous studies. 22 Although mitochondrial DNA is mostly unmethylated (mean methylation 6.43% Figure 1a), CpG methylation in the human and EBV genome is comparably distributed, although the EBV genome exhibits hardly any fully methylated sites (Figure 1a). Overall, EBV shows a high level of DNA methylation (mean methylation 80.18%), as it was previously shown for BL cell lines.…”

Head and neck lesions of Kimura's disease: Exclusion of human herpesvirus-8 and Epstein-Barr virus by in situ hybridisation and polymerase chain reaction. An immunohistochemical study

“…DNA sequences were determined by nested PCR using primers specific for a 233-bp-long fragment of HHV-8 (ORF 26) and a 269-bp-long fragment of EBV. Moreover, the presence of EBV DNA was analysed by in situ hybridisation using the technique of Leenman et al (2004).…”