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This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.
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.
The Epstein-Barr virus (EBV) is the first human tumor virus to have been isolated1. EBV, formally referred to as Human herpesvirus 4 (HHV-4)2, is part of the gamma herpes virus subfamily of the herpes virus family and is the prototype of the Lymphocryptovirus genus. Nearly 90-95% of the world's adult population is infected by the virus3. In most cases, initial infection occurs within the first 3 years of life and is asymptomatic, however, if infection occurs later during adolescence, it may give rise to an illness referred to as infectious mononucleosis4. EBV is able to infect resting B cells inducing them to become proliferative B lymphoblasts in which the virus establishes and maintains a latently infected state5. EBV can reactivate at any time and thus lead to recurrent infections6.
Over the past 50 years, the association between some viruses and the development of human malignancies has become increasingly apparent, and today it is estimated that 15% to 20% of all human cancers are related to viral infections7. The herpes viruses, including EBV, are some of the best studied examples of these types of tumor viruses8. In fact, EBV can cause many types of human malignancies, such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), diffuse large B cell lymphoma, and lymphoproliferative diseases in immunocompromised hosts9,10. EBV has also been shown to be associated with the development of systemic autoimmune diseases. Some examples of these autoimmune disorders are rheumatoid arthritis (RA), polymyositis-dermatomyositis (PM-DM), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and Sjögren's syndrome (SS)11. EBV is also associated with the development of inflammatory bowel disease (IBD)12.
Many of these diseases can be studied or modeled using cell culture, mice, or other organisms that are infected with EBV. That is why EBV particles are needed to infect cells or organisms, whether in in vitro or in vivo models13,14,15,16, hence the need to develop a technique that allows isolation of viral particles at a low cost. The protocol described here provides guidelines for an easy way to reliably isolate EBV particles from a relatively accessible cell line and to quantitate the particles using real-time PCR, which is cost-effective and readily available to most laboratories. This is in comparison to several other methods that have been described to isolate EBV from different cell lines17,18,19,20.
P3HR-1 is a BL cell line that grows in suspension and is latently infected with an EBV type 2 strain. This cell line is an EBV producer and can be induced to produce viral particles. The goal of this manuscript is to showcase a method that permits the isolation of EBV particles from the P3HR-1 cell line, followed by quantification of the viral stock that could later be used for both in vitro and in vivo EBV experimental models.
NOTE: EBV should be considered a potentially biohazardous material, and thus should be handled under Biosafety Level 2 containment or higher. A lab coat as well as gloves should be worn. If there is potential for exposure to splashes, eye protection should also be considered. The following procedure should be conducted in a Biological Safety Cabinet.
1. Counting the P3HR1 cells
Figure 1: Cell counting using a hemocytometer chamber. Four quadrants are counted using a light microscope; these quadrants are represented in green. Total Cells Counted in the formula indicated in step 1.3.3 of the protocol described here is the sum of the number of cells in quadrants 1, 2, 3 and 4. Cells indicated in blue should be counted, while cells in red should not be counted since they are touching the top, right, bottom, or left borders of the quadrant Please click here to view a larger version of this figure.
2. Preparing the plate for culture
3. Induction and isolation of Epstein-Barr virus particles
4. DNA extraction from viral particles
CAUTION: Extreme care should be taken when handling phenol, as it is toxic and corrosive and has the ability to cause severe burns. Phenol is light sensitive and oxidizes upon contact with light or air. Store it in a light-resistant container or alternatively cover the phenol tube with an opaque material like aluminum foil.
5. Checking DNA concentration and purity
6. Quantification by real-time polymerase chain reaction
7. Checking for biological activity/infectivity of the viral particles
The goal of this procedure is to isolate EBV particles in a suspension with known viral titer, that could subsequently be used to model EBV infection. Thus, it is of utmost importance to use optimal concentrations of the different reagents to obtain the highest EBV yield out of the procedure.
An optimization trial was performed to determine the concentrations of PMA and DMSO that would yield the highest number of EBV particles (Figure 2). A DMSO concentration of 0...
The production of EBV particles is necessary for understanding the biology of this virus as well as its associated diseases. Here we described the production of these particles from the P3HR-1 cell line. This cell line is not the only EBV-producer line; in fact, EBV particles have also been isolated from B95-8 cells21,22 as well as the Raji cell line18,19. The EBV lytic cycle has been induced in these cel...
The authors declare no conflicts of interest.
Funding for this work was supported by grants to ER from the Asmar Research Fund, the Lebanese National Council for Scientific Research (L-CNRS), and the Medical Practice Plan (MPP) at the American University of Beirut.
Name | Company | Catalog Number | Comments |
0.2 mL thin-walled PCR tubes | Thermo Scientific | AB0620 | Should be autoclaved before use |
0.2-10 µL Microvolume Filter Tips | Corning | 4807 | Should be autoclaved before use |
0.5-10 µL Pipette | BrandTech | 704770 | |
10 mL Disposable Serological Pipette | Corning | 4488 | |
1000 µL Filtered Pipette Tips | QSP | TF-112-1000-Q | |
100-1000 µL Pipette | Eppendorf | 3123000063 | |
100x20 mm Cuture Plates | Sarstedt | 83.1802 | |
10-100 µL Pipette | BrandTech | 704774 | |
15 mL Conical Tubes | Corning | 430791 | |
200 µL Filtered Pipette Tips | QSP | TF-108-200-Q | |
20-200 µL Pipette | Eppendorf | 3123000055 | |
50 mL Conical Tubes | Corning | 430828 | |
CFX96 Real-Time C-1000 Thermal Cycler | Bio-Rad | 184-1000 | |
DMSO | Amresco | 0231 | |
DNase/RNase Free Water | Zymo Research | W1001-1 | |
EBER Primers | Macrogen | N/A | Custom Made Primers |
EBV DNA Control (Standards) | Vircell | MBC065 | |
Ethanol (Laboratory Reagent Grade) | Fischer Chemical | E/0600DF/17 | |
Fetal Bovine Serum | Sigma | F9665 | |
Fresco 21 MicroCentrifuge | Thermo Scientific | 10651805 | |
Glycogen Solution | Qiagen | 158930 | |
Hemocytometer | BOECO | BOE 01 | |
Inverted Light Microscope | Zeiss | Axiovert 25 | |
iTaq Universal SYBR Green Supermix | Bio-Rad | 172-5121 | |
Microcentrifuge Tube | Costar (Corning) | 3621 | Should be autoclaved before use |
P3HR-1 Cell Line | ATCC | HTB-62 | |
Penicillin-Streptomycin Solution | Biowest | L0022 | |
Phenol | VWR | 20599.297 | |
Phorbol 12-myristate 13-acetate (PMA) | Sigma-Aldrich | P8139 | |
Pipette Filler | Thermo Scientific | 9501 | |
Precision Wipes | Kimtech | 7552 | |
RPMI-1640 Culture Medium | Sigma | R7388 | |
SL 16R Centrifuge | Thermo Scientific | 75004030 | |
Sodium Acetate | Riedel-de Haën (Honeywell) | 25022 | |
Spectrophotomer | DeNovix | DS-11 | |
Tris-HCl | Sigma | T-3253 | |
Trypan Blue Solution | Sigma | T8154 | |
Water Jacketed CO2 Incubator | Thermo Scientific | 4121 |
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