One-step multiplex real-time PCR assay to analyse the latency patterns of Epstein-Barr virus infection

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Abstract

Epstein-Barr virus (EBV) establishes a latent infection with three types of viral gene expression. These latency types can be distinguished by the expression patterns of EBV nuclear antigen (EBNA)1, EBNA2, latent membrane protein (LMP)1, and LMP2. The EBV lytic cycle is initiated by the transcription of the EBV immediate early BZLF1 gene, which can be used to distinguish between a latent and a lytic infection. In this study, a one-step multiplex real-time PCR assay was developed to quantify the EBNA1, EBNA2, LMP1, LMP2, and BZLF1 expression levels simultaneously by relative quantification. To validate this assay, the quantitation of viral gene transcription was performed in EBV-positive B, T, and natural killer cell lines. Because of its rapidity, sensitivity, and specificity, this new assay can be used for quantitative analyses of the latency patterns of EBV infection and the switch from latency to lytic viral replication.

Introduction

Epstein-Barr virus (EBV) is a species in the genus Lymphocryptovirus, subfamily Gammaherpesvirinae, family Herpesviridae. EBV is a ubiquitous virus that infects most individuals by early adulthood. In primary human infection, cell-free EBV in saliva infects naive B lymphocytes, causing them to become proliferating blasts (Thorley-Lawson and Gross, 2004). It then establishes a latent infection in those lymphocytes, which are largely nonpermissive for virus replication (Niedobitek et al., 1989, Tierney et al., 1994). In immunocompromised hosts, EBV is frequently reactivated and can result in symptomatic diseases. Several malignancies, including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disorders, are related to EBV infection (Rickinson and Kieff, 2001). The clonal expansion of EBV-infected T cells and natural killer (NK) cells is also indicated to play a central role in the pathogenesis of chronic active EBV infection (Kimura, 2006, Kimura et al., 2001).

Viral gene expression in EBV-associated diseases is limited to one of three latency patterns (Rickinson and Kieff, 2001). In latency type I, which is found in Burkitt's lymphoma (Tao et al., 1998), only EBV nuclear antigen (EBNA)1 and EBV-encoded small RNAs (EBERs) are expressed. In latency type II, which is characteristic of Hodgkin's disease (Deacon et al., 1993) and nasopharyngeal carcinoma (Brooks et al., 1992), EBNA1, latent membrane protein (LMP)1, LMP2, and EBERs are expressed. In latency type III, which is associated with lymphoproliferative disorders (Young et al., 1989), all of the latency genes (EBNA1, EBNA2, EBNA3a-c, EBNA-LP, LMP1, LMP2, and EBERs) are expressed. The EBV lytic cycle is initiated by the transcription of the EBV immediate early BZLF1 gene, whose product, the ZEBRA protein, is an essential factor in the switch from latency to lytic virus replication (Chevallier-Greco et al., 1986, Takada et al., 1986). Therefore, as a hallmark of lytic infection, BZLF1 is used to distinguish between latent and lytic infections.

To identify the latency type of EBV-infected cells, the pattern of viral gene expression has been analysed using various techniques (Rickinson and Kieff, 2001). Reverse transcription (RT)-PCR is a sensitive method that has been used widely to detect EBV gene expression (Kerr et al., 1992, Tierney et al., 1994); however, it is qualitative and time- and labor-intensive. Furthermore, little is known about the quantitative transcription levels of EBV latent genes in virus-infected cells, while qualitative analyses have been published extensively. Quantitative differences in EBV gene expression could be related to the pathogenesis of EBV-associated diseases, and they may provide meaningful information about prognosis and therapeutic intervention.

In this study, a one-step multiplex real-time PCR assay was developed and validated to quantify the relative expression of EBV latent and lytic genes. This method allows a rapid, sensitive, and specific analysis of the latency patterns of EBV infection.

Section snippets

Cell lines

LCL, a lymphoblastoid cell line transformed with B95-8 EBV, was used as a positive control. BJAB (Menezes et al., 1975), an EBV-negative B cell line, was used as a negative control. The EBV-positive Burkitt's lymphoma cell lines used include Akata (Takada et al., 1991), Raji (Pulvertaft, 1965), Daudi (Nadkarni et al., 1969), and Namalwa (Henderson et al., 1983). The EBV-positive T cell lines used include SNT-8, -13, -15, and -16 (Zhang et al., 2003). The EBV-positive NK cell lines used include

Detection of EBV gene expression by one-step multiplex real-time PCR

To determine the optimal concentrations of the primers and probes, various concentrations of the primer and probe sets were evaluated by one-step multiplex real-time PCR assay. Serial dilutions of LCL total RNA were tested, and standard curves were constructed for each gene from the cycle of threshold values. The optimal concentration of each primer and probe was 200 and 100 nM, respectively. Gene expression was not detected in the reverse transcriptase-negative samples, indicating that the

Discussion

Nonquantitative methods such as qualitative RT-PCR have been used to identify the latency pattern of EBV infection (Kerr et al., 1992, Tierney et al., 1994); however, a quantitative and more rapid method is required to analyse latent and lytic viral gene expression. In this study, a one-step multiplex real-time PCR assay was developed that simultaneously detects the expression of four latency genes (EBNA1, EBNA2, LMP1, and LMP2), one lytic gene (BZLF1), and one housekeeping gene (B2M) in

Acknowledgments

We thank Tatsuya Tsurumi (Aichi Cancer Center) for the Raji, Akata, and Daudi cell lines; Takeshi Sairenji (Tottori University) for the Namalwa cell line; and Norio Shimizu (Tokyo Medical and Dental University) and Ayako Demachi-Okamura (Aichi Cancer Center) for the SNK-1, -6, and -10 and the SNT-8, -13, -15, and -16 cell lines.

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