Journal of General Virology (2000), 81, 2417–2423. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Expression of Epstein–Barr virus lytic gene BRLF1 in nasopharyngeal carcinoma : potential use in diagnosis Ping Feng, 1 Ee Chee Ren, 1 Dingxiang Liu, 2 Soh Ha Chan 1 and Huaizhong Hu 1 Department of Microbiology, Faculty of Medicine 1 and Institute of Molecular Agrobiology 2 , National University of Singapore, Block MD4/4A, 5 Science Drive 2, Singapore 117597, Republic of Singapore Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein– Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT–PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83–0%), but only in 1 of 53 control samples (1–9 %). Furthermore, the antibody binding regions were found in the C-terminal two- thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC. Introduction Epstein–Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects more than 90 % of the human population. The primary infection is generally asymptomatic or causes in- fectious mononucleosis in some circumstances (Rickinson & Kieff, 1996). A lifelong viral persistence is established as a form of in vivo latency in healthy carriers following primary infection, with low copies of episomal virus maintained in resting memory B cells (Babcock et al., 1998 ; Tierney et al., 1994 ; Anagnostopoulos et al., 1995). Spontaneous reactivation of the latent infection can occur in oropharyngeal epithelium (Sixbey et al., 1984) and}or local mucosal lymphoid tissues (Babcock et al., 1998 ; Anagnostopoulos et al., 1995), possibly by responding to physiological signals such as cytokines, steroid hormones and CD40 ligand (Thorley-Lawson et al., 1996). Disruption of latency in EBV-transformed lympho- blastoid cells in vitro can be induced by treatment with various Author for correspondence : Huaizhong Hu. Fax ›65 776 6872. e-mail michuhz!nus.edu.sg chemicals, including phorbol esters, n-butyrate, calcium iono- phores and anti-immunoglobulin (Rickinson & Kieff, 1996 ; Thorley-Lawson et al., 1996). EBV utilizes a variety of distinct genetic programmes, each of which is categorized by ex- pression of a unique set of viral genes, at various stages of its life-cycle. These programmes can be grouped into either the latency-associated programme or the replicative programme (Rodriguez et al., 1999). EBV is associated with a spectrum of malignancies of lymphoid and epithelial cell origin (Rickinson & Kieff, 1996), such as Burkitt’s lymphoma, T-cell lymphoma, Hodgkin’s disease, undifferentiated nasopharyngeal carcinoma (NPC) and gastric carcinoma. NPC is the most consistently EBV- associated malignancy and occurs mainly among the Chinese population in Southern China and Southeast Asia. However, the EBV genome has been detected in malignant epithelial cells in NPC patients regardless of geographical origin (Wolf et al., 1973). The demonstration of monoclonality of the viral DNA indicates that the malignancy has arisen from clonal expansion of a single EBV-infected progenitor cell (Raab-Traub & Flynn, 1986). EBV latency-associated gene expression is consistently 0001-7190 # 2000 SGM CEBH