Nucleic acid sequence-based amplification, a new method for analysis of spliced and unspliced Epstein-Barr virus latent transcripts, and its comparison with reverse transcriptase PCR.
Nucleic acid sequence-based amplification (NASBA) assays were developed for direct detection of Epstein-Barr virus (EBV) transcripts encoding EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and BamHIA rightward frame 1 (BARF1) and for the noncoding EBV early RNA 1 (EBER1). The sensitivities of all NASBAs were at least 100 copies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA assays. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clinical material. BARF1 expression was found in 6 of 7 nasopharyngeal carcinomas (NPC) but in 0 of 22 Hodgkin's disease (HD) cases, whereas LMP2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For detection of EBNA1 transcripts in HLs (n = 12) and T- and B-cell non-Hodgkin's lymphomas (n = 3 and n = 2, respectively), NASBA was compared with reverse transcriptase (RT) PCR. Two samples were positive only with NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We conclude that NASBA is suitable for sensitive and specific detection of the above-mentioned EBV transcripts, regardless of their splicing patterns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs developed in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.
Brink
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AA
Section Molecular Pathology, Department of Pathology, University Hospital Vrije Universiteit, 1007 MB Amsterdam, The Netherlands.
Vervoort
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MB
Middeldorp
J M
JM
Meijer
C J
CJ
van den Brule
A J
AJ
D001483:Base Sequence; D002461:Cell Line, Transformed; D017931:DNA Primers / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D006689:Hodgkin Disease / Q000821:virology; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000821:virology; D009303:Nasopharyngeal Neoplasms / Q000821:virology; D021141:Nucleic Acid Amplification Techniques; D012326:RNA Splicing; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014763:Viral Matrix Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
C086495:BARF1 protein, Human herpesvirus 4; D017931:DNA Primers; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; C099537:Epstein-Barr virus encoded RNA 1; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D014764:Viral Proteins