Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

Abstract

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world's adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles. Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 °C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation. © 2022 JoVE Journal of Visualized Experiments.