Visual Detection and Evaluation of Latent and Lytic Gene Expression during Epstein-Barr Virus Infection Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

Xiaoying Liu,Jingfeng Tang,Man Wang,Yefu Wang,Qiang Ma

doi:10.3390/ijms141223922

Abstract

Epstein-Barr virus (EBV)-associated disease exhibits distinct gene expression patterns characterized by the transcription of EBV nuclear antigen (EBNA) 1, EBNA2, latent membrane protein (LMP) 1, LMP2A, and BZLF1 (Zebra). A series of visual reverse transcript loop-mediated isothermal amplification (RT-LAMP) assays were performed to examine the expression of EBNA1, EBNA2, LMP1, LMP2A and BZLF1. The sensitivity of RT-LAMP for these transcripts was approximately equivalent to real-time RT-PCR (RT-qPCR), which was developed to quantify relative levels of EBV transcripts, and 10 to 100-fold more sensitive than conventional RT-PCR. Cross-reactions to other viruses were not observed upon examination of cell lines infected with herpes simplex viruses-1 and -2 (HSV-1 and -2), varicella zoster virus (VZV), human cytomegalovirus (HCMV) or Kaposi’s sarcoma-associated herpesvirus. When applied to 146 specimens, RT-LAMP exhibited high clinical sensitivity and specificity, with an excellent agreement (κ > 0.92) compared to RT-qPCR. These assays are convenient for rapid early diagnosis and for surveillance of EBV-infected individuals by evaluating the EBV transcriptional profile, because the results can be visualized with the naked eye. These assays may be employed in further investigations because they can aid the design of improved therapeutic regimens and can be used specifically in resource-poor settings.

Highlights

Epstein-Barr virus (EBV) infects more than 95% of people and can lead to life-long, latent infection by attacking the host immune system in immunocompetent individuals [1]
We reported a technique termed loop-mediated isothermal amplification (LAMP) to resolve the problems that low sensitivity, false-positives, or the requirements of expensive equipment and skilled operation [19]
The only equipment needed for reverse transcript loop-mediated isothermal amplification (RT-LAMP) is a cost-effective laboratory water bath or a heat block that supplies a constant temperature of 63 °C

Summary

Introduction

Epstein-Barr virus (EBV) infects more than 95% of people and can lead to life-long, latent infection by attacking the host immune system in immunocompetent individuals [1]. Primary EBV infection usually remains asymptomatic in young children and displays as acute infectious mononucleosis (IM) in. Young adults tend to recover without sequelae [2,3]. Latent EBV infection is involved in the multistep pathogenesis of many malignancies, including lymphoid neoplasms (Hodgkin’s lymphoma (HL), Burkitt’s lymphoma (BL), EBV-associated post-transplantation lymphoproliferative disorders (PTLD), senile EBV-associated B-cell lymphoproliferative disorder and. T/NK-cell lymphomas) and epithelial carcinomas (nasopharyngeal carcinoma (NPC), gastric and oral cancers) [5,6,7]. At least four basic programs (including types I, II, III and 0) of virus latency can been identified on the basis of differential gene expression patterns in different EBV-associated disorders

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