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

Abstract

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.

FIG. 1

Schematic representations of different types of EBV transcripts and localization of NASBA primers. (a) Transcript which is spliced in the noncoding but not the coding domain, like EBNA1 transcripts (10, 14, 21). All possible coding transcripts are detected by NASBA with primers localized within the ORF. (b) Transcript which is spliced in the coding domain, like LMP1 and LMP2 (12, 13). (c) Transcript which is not spliced at all, like EBER1 (6). (d) Transcript whose splicing pattern is not known, like BARF1 (27). Although the splicing patterns of the BamHIA region are highly complex, the selection of NASBA primers within the ORF of interest provides reliable detection without false negatives. Large open bars represent exons; small black bars represent NASBA primers. Abbreviations: ATG, start codon; AAAA, poly(A) tail; BKRF1, BamHIK rightward frame 1.

FIG. 2

(a) EBNA1 NASBA analysis of JY RNA with the four different primer combinations; combinations 1.1-2.1 and 1.2-2.1 clearly give stronger signals than the other two combinations. (b) Determination of the relative sensitivity of primer set 1.2-2.1. A positive signal was obtained even when an amount of RNA equivalent to only one JY cell was present. The number of JY cells is indicated above each lane. −, negative water control; +, positive control.

FIG. 3

Relative sensitivities of the LMP1 (a), LMP2 (b) (left, primer set 1.1-2.2 [11/22]; right, primer set 1.2-2.1 [12/21]), and EBER (c) (left, RNAzol isolation; right, silica-based isolation) NASBAs and analytical sensitivity of the BARF1 NASBA (d). The relative sensitivity of the LMP1 and LMP2 NASBAs is 1 JY cell, and the analytical sensitivity of the BARF1 NASBA is 10 copies of cRNA; due to photographic reduction, these signals may not be visible. The relative sensitivity of the EBER NASBA after RNAzol isolation is 100 JY cells. The number of JY cells, and for BARF1, the number of cRNA copies, is indicated above each lane. +, positive control; −, negative control.

FIG. 4

Evaluation of the LMP2 (a) and BARF1 (b) NASBAs on NPC and HD samples. LMP2 signal is observed in all NPC cases and in 17 of 22 HD cases, whereas BARF1 signals are only found in the NPC material (in 6 of 7 cases).