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Analysis of Epstein–Barr virus latent gene expression in endemic Burkitt's lymphoma and nasopharyngeal carcinoma tumour cells by using quantitative real-time PCR assays

¹ Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
² Department of Clinical Oncology, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong

Correspondence
Andrew I. Bell
a.i.bell{at}bham.ac.uk

Studies of Epstein–Barr virus (EBV)-positive cell lines have identified several forms of virus latency, but the patterns of virus gene expression in EBV-positive tumour cells appear more variable. However, it is unclear to what extent these differences merely reflect the increased sensitivities of different detection methods. Here, the design and validation of novel real-time RT-PCR assays to quantify relative levels of EBV transcripts are described. When the new assays were used to screen a collection of endemic Burkitt's lymphoma tumours, abundant Qp-driven EBNA1 expression was found, whereas the other latent transcripts (with the exception of LMP2A) were either absent or detectable only at trace levels. Analysis of 12 nasopharyngeal carcinoma biopsies revealed significant levels of EBNA1 and LMP2A transcripts in almost every case but, in contrast to previous reports, LMP1 expression was undetectable. These new quantitative assays may help to provide a clearer picture of EBV gene expression in tumour material.

Supplementary methods, a sequence alignment of LMP1 exons 2 and 3, figures showing detection of EBV transcripts by real-time RT-PCR and conventional end-point RT-PCR and a table showing TaqMan primer/probe combinations used to detect EBV transcripts are available in JGV Online.

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