Epstein-Barr virus diagnostic and therapeutic opportunities in nasopharyngeal carcinoma

Middeldorp, J.M. [Promotor]Pegtel, D.M. [Copromotor

Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers

Natural variation of Epstein-Barr virus genes, proteins and pri-microRNA

Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains including many primary isolates have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains named QCIGP results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through SNPs in the pri-miRNA outside of the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future more directed analysis of association of specific EBV variation with EBV biology and EBV associated diseases.IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Relationships between EBV genome sequence variation and health, disease, geography and ethnicity of the host may thus be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focussing on variation in LMP1, Zp, gp350, EBNA1 and the BART miRNA cluster 2, new relationships to the known type 1/type 2 strains are demonstrated and novel classification of EBNA1 and the BART miRNAs is proposed