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 0.8% and a PMA concentration of 35 ng/µL were optimal and resulted in high EBV concentrations. The concentration of viral particles obtained from the optimization protocol was 917,471 viral particles/µL.
Figure 2: EBV DNA copies/mL obtained upon induction of P3HR-1 cells with phorbol 12-myristate 13-acetate (PMA). P3HR-1 cells (105 cells) were cultured alone or induced with different concentrations of PMA and DMSO using the protocol described here. The error bars indicate the standard deviation. A Student's t-test was performed comparing PMA/PMA and DMSO-treated cells to control cells; * indicates p < 0.05. Please click here to view a larger version of this figure.
For this protocol to be considered effective, step 6 should reveal a reasonable concentration of viral particles, and step 7 should show that these viral particles are indeed infectious. The BC-3 cell line used in step 7 is EBV-negative, so upon quantification of the EBV genome copy numbers in negative controls (that were not subjected to this protocol), no EBV DNA should be detected.
However, EBV DNA should be detected in both the cells and the culture medium of the experimental wells (that were subjected to this protocol), for the following reasons: EBV that infects BC-3 cells will replicate inside these cells, accounting for the fact that EBV DNA will be detected in the cell pellet and upon replication, BC-3 cells will shed viral particles to the outside; thus, EBV DNA will be detected in the culture medium supernatant (after DNA extraction).
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 cells with n-butyrate. Alternatively, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), also known as phorbol 12-myristate-13-acetate (PMA), can be used. Overall, the protocol detailed here can be used employing these cells instead of the P3HR-1 line. However, the B95-8 line was derived from a cotton-top tamarin (Saguinus oedipus) that is currently classified as a critically endangered primate species23; hence, this line is rarely sold by vendors with restrictions on its sale and shipping. A license is also required by the convention on the international trade in endangered species of wild fauna and flora (CITES) for all orders outside the United Kingdom. While the Raji cell line is commercially available, the synthesis of replicative viral DNA and the production of viral capsid antigen (VCA) do not occur in these cells after induction by chemicals24,25. However, these can be readily induced by complementation after superinfection with EBV isolated from P3HR-1 cells20.
Due to the absence of a direct plaque assay for the enumeration of EBV particles26, we relied on a real-time polymerase-chain reaction. There are other methods of quantification that may work equally well for our purpose, such as immunofluorescence or PCR employing fluorescent probes that may offer enhanced specificity. The choice of real-time PCR using genome copy standards is based on the fact that this is readily accessible to most laboratories, less laborious, less technically challenging, and more cost-efficient27. Hence, other methods of quantification could work equally well and may be used based on availability and experience.
One of the most critical steps in the protocol detailed here is the plating of cells in step 2.1 and seeding with a cell number within the range recommended for the size of the plate. A low number of cells yields a low concentration of viral particles, while a large number of cells would crowd the plate, inhibit the growth and replication of cells, and hence slow the lytic cycle of the virus. This is why extreme care should be taken while counting the cells in step 1, calculating the required volume of suspension A, and finally plating the cells in step 2.
A limitation of the protocol is the assumption that one copy of the EBV genome corresponds to one viral particle. While this assumption is plausible, whether such particles are defective or incomplete cannot be detected using this assay. Since there are no plaque formation assays for EBV as there are for many other viruses, DNA extraction followed by real-time PCR remains an accepted method to quantify the viral preparation. Another limitation is that the virus is induced from a continuous mammalian cell line that may accumulate mutations upon many rounds of passaging. Such mutations may affect the virus itself, resulting possibly in altered in vivo or ex vivo properties of the virus. A potential solution would be to regularly sequence the cellular and viral genome. Alternatively, cells can be discarded after a few passages and the culture can be restarted anew from a liquid nitrogen-stored cell sample with a low number of previous passages. It is also worth noting that the EBV particles obtained from the P3HR-1 cell line lack the EBNA2 antigen, which is usually expressed in B lymphocytes that are latently infected with EBV, as part of six viral nuclear proteins in total28. The EBNA2 protein activates the promoters of genes that express proteins associated with the type III of EBV latency. However, recent studies have shown that EBNA2-deficient EBV is able to form tumors in CBH mice, and that the P3HR-1 cell line hence remains a useful model of EBV positive lymphomas29. On the other hand, EBV from P3HR-1 cells can be used to study the roles played by EBNA2 while using an EBNA2-positive EBV as control. In summary, EBV obtained from a continuous P3HR-1 cell line has a significant research value; it can be used to study the biology, infectivity, virulence, as well as many other properties of EBV.
The authors have nothing to disclose.
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.
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 | |
100×20 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 |