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.

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
<ol>
	<li>Centrifuging and resuspending cells
	<ol>
		<li>Transfer the cell suspension from a 100 mm culture plate (or T-25 flask) of an ongoing P3HR1 cell culture at 80% confluency to a 15 mL conical tube. The seeding density of P3HR-1 cells is 1 x 106 cells/mL. Maintain in complete RPMI culture medium (79% RPMI culture medium, 20% Fetal Bovine Serum, 1% Pen-Strep antibiotic) at 37 &#176;C and 5% CO2, and passage every 3-4 days.</li>
		<li>Centrifuge for 8 min at 120 x g. After centrifugation, discard the supernatant, resuspend the cell pellet in 1 mL of complete RPMI culture medium, and mix well (this solution will be referred to as suspension A).</li>
	</ol>
	</li>
	<li>Preparing the cell suspension for counting
	<ol>
		<li>Prepare a diluted cell suspension B by mixing 2 &#181;L of cell suspension A with 8 &#181;L of culture medium. Add 10 &#181;L of 0.4% trypan blue to suspension B to obtain suspension C. Mix the preparation well by gentle pipetting.</li>
	</ol>
	</li>
	<li>Counting cells using a hemocytometer
	<ol>
		<li>Place a glass coverslip on the hemocytometer and load 10 &#181;L of suspension C. Place the hemocytometer under a light microscope and count the number of cells that are not stained blue in each of the four quadrants of the hemocytometer chamber using the 40x microscope objective (Figure 1).</li>
		<li>Trypan blue stains dead cells blue; do not count these cells, nor cells that are touching any of the top, bottom, right, or left borders of each hemocytometer quadrant.</li>
		<li>Calculate the concentration of cells per mL using the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/64279/64279eq01.jpg" style="margin: auto;" /> 
		NOTE: The number of quadrants counted is four, and 10-4 mL is the volume of the squares on the hemocytometer. The four quadrants which should be counted are represented in green in Figure 1. Total Cells Counted in the formula is the sum of the numbers of cells in quadrants 1, 2, 3, and 4. Cells represented in blue in Figure 1 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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64279/64279fig01.jpg" /> 
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 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64279/64279fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparing the plate for culture
<ol>
	<li>Place a volume of cell suspension A in a 15 mL conical tube. The volume required is dependent on the cell count. Adjust the volume such that the number of cells is roughly 2.2 x 106 cells for a 100 mm culture plate.</li>
	<li>Add 5 mL of complete culture medium to the tube and then transfer the contents of the tube to a 100 mm culture plate. Add 80 &#181;L of dimethyl sulfoxide (DMSO) to the culture plate. 
	NOTE: DMSO is light sensitive, hence it should be stored in a light-resistant container or covered with an opaque material like aluminum foil.</li>
	<li>Add 350 &#181;L of 1 mg/mL phorbol 12-myristate 13-acetate (PMA) to the tube. The final concentration of PMA should be 35 ng/mL, and that of DMSO should be 0.08%. 
	CAUTION: PMA is toxic, corrosive, and carcinogenic, hence it should be handled with extreme care. PMA is light sensitive, hence it should be stored in a light-resistant container or covered with an opaque material like aluminum foil.</li>
	<li>Add 4.27 mL of culture medium so that the total volume is 10 mL. Mix the contents of the tube by tilting, then transfer the contents to a 100 mm culture plate. Leave the plate in a cell culture incubator for 5 days at 37 &#176;C, 5% CO2.</li>
</ol>
3. Induction and isolation of Epstein-Barr virus particles
<ol>
	<li>After 5 days in the incubator, centrifuge the contents of the plate at 120 x g for 8 min to pellet the cells. Collect the cell-free, virus-containing supernatant and discard the cell pellet.</li>
	<li>Centrifuge the supernatant at 16,000 x g for 90 min at 4 &#176;C to pellet the virus particles. Discard the supernatant and resuspend the virus pellet in 5 mL of culture medium. 
	NOTE: Phosphate-buffered saline (PBS) can be used to resuspend the virus pellet instead of culture medium.</li>
	<li>Aliquot the viral suspension obtained into 20 tubes, each containing 250 &#181;L of the viral suspension. Store the suspension at -80 &#176;C.</li>
</ol>
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.
<ol>
	<li>Separation of proteins from DNA
	<ol>
		<li>Add 500 &#181;L of TrisCl-saturated phenol to one of the 250 &#181;L tubes. Add 100 &#181;L of water (to increase the volume of the aqueous phase) and vortex well to obtain a pink emulsion. Centrifuge for 15 min at 9650 x g.</li>
	</ol>
	</li>
	<li>DNA precipitation
	<ol>
		<li>Collect the supernatant (transparent, aqueous phase) and transfer it to a new 1.5 mL microcentrifuge tube.</li>
		<li>Add an equivalent of 1/10 of the supernatant&#39;s volume of cold sodium acetate (3 M, pH 5.2) and mix by pipetting up and down. Add 1 &#181;L of 20 mg/mL glycogen and mix by pipetting. 
		NOTE: This step is optional, but the addition of glycogen enhances DNA precipitation as well as makes it easier to visualize the DNA pellet.</li>
		<li>Add three times the supernatant&#39;s volume of cold 100% ethanol. Store at -80 &#176;C overnight. 
		NOTE: The procedure can be stopped here to be resumed at a later time. The sample can be stored at -80 &#176;C for 1 h, or alternatively overnight. Overnight storage enhances DNA precipitation and is thus recommended.</li>
	</ol>
	</li>
	<li>Isolating the viral DNA
	<ol>
		<li>The following day, centrifuge at 9650 x g for 15 min at 4 &#176;C and discard the supernatant to obtain a DNA pellet.</li>
		<li>Wash the pellet three times with 1 mL of cold 70% ethanol, centrifuge at 9650 x g for 15 min, and discard the supernatant.</li>
		<li>Air-dry the pellet for about 10 min. Resuspend the pellet in 10-50 &#181;L of nuclease-free distilled water (depending on the size of the DNA pellet).</li>
		<li>Store the samples at -20 &#176;C for later processing or at 4 &#176;C overnight to ensure maximal DNA dissolution followed by storage at -20 &#176;C. Alternatively, directly proceed with step 5.</li>
	</ol>
	</li>
</ol>
5. Checking DNA concentration and purity
<ol>
	<li>After cleaning the pedestal of a microspectrophotometer with a delicate task wiper, load 1 &#181;L of the DNA preparation.</li>
	<li>Note the concentration of the DNA in the sample. Check the ratios of absorbance at both <img alt="Equation 2" src="/files/ftp_upload/64279/64279eq02.jpg" style="margin: auto;" /> and <img alt="Equation 3" src="/files/ftp_upload/64279/64279eq03.jpg" style="margin: auto;" />. DNA will absorb at 260 nm, proteins at 280 nm, and organic substances such as phenol will absorb at 230 nm. A ratio of 1.8-2 is considered sufficient for real-time PCR.</li>
</ol>
6. Quantification by real-time polymerase chain reaction
<ol>
	<li>Preparation of the PCR reaction mixes
	<ol>
		<li>Place 5 &#181;L of a SYBR green real-time PCR mix in 0.2 mL PCR tubes. Add to each tube 1 &#181;L of 7.5 pmol/&#181;L forward primer and 1 &#181;L of 7.5 pmol/&#181;L reverse primer. Forward primer sequence: 5&#39;-CCCTAGTGGTTTCGGACACA-3&#39;; reverse primer sequence: 5&#39;-ACTTGCAAATGCTCTAGGCG-3&#39;. The gene amplified to determine the EBV genome copy number is the Epstein-Barr virus small RNA 2 (EBER-2) gene.</li>
		<li>To one of the tubes, add 2 &#181;L of nuclease-free water and 1 &#181;L of viral DNA from step 4. The total volume of the reaction mixture is 10 &#181;L. 
		NOTE: Depending on the concentration of DNA in the sample, a dilution step may be needed. The dilution factor F should be noted and will be integrated in the formula in step 6.3.</li>
		<li>Prepare other tubes that will be used as standards with known EBV genome copy numbers (1000, 2000, 5000, 10,000, and 54,000 copies).</li>
	</ol>
	</li>
	<li>Run the PCR mixtures starting with an initial step of activation at 95 &#176;C for 5 min, then 40 cycles at 95 &#176;C and 58 &#176;C (annealing) for 15 s and 30 s, respectively.</li>
	<li>Generate a qPCR standard curve by plotting the Ct values of the standards against the log of the number of EBV genome copies per standard tube. Employing the standard curve plot equation, derive the number of EBV genome copies in the PCR tube containing the induced viral DNA. Then, use the following formula to calculate the concentration of the induced viral preparation: 
	<img alt="Equation 4" src="/files/ftp_upload/64279/64279eq04.jpg" style="margin: auto;" /> 
	Where X is the number of EBV genome copies derived from the standard curve and F is the dilution factor used for setting up the DNA utilized per PCR reaction.</li>
</ol>
7. Checking for biological activity/infectivity of the viral particles
<ol>
	<li>Transfer BC-3 cells from a 100 mm culture plate at 80% confluency to a 15 mL conical tube. The seeding density of P3HR-1 cells is 1 x 106 cells/mL. Maintain in complete RPMI culture medium (79% RPMI culture medium, 20% Fetal Bovine Serum, 1% Pen-Strep antibiotic) at 37 &#176;C and 5% CO2, and passage every 3-4 days.</li>
	<li>Count the BC-3 cells the same way the P3HR-1 cells were counted by following step 1. To a 96-well plate, add 105 BC-3 cells to each well. Use three, six, nine, or 12 wells to ensure the significance of results and minimize errors.</li>
	<li>Add a volume of viral stock to each well, depending on the concentration of the viral preparation determined in step 6.3. The MOI can vary, and optimization of this protocol will reveal the best MOI for infection (an MOI range of 2-50 is recommended). Ensure the total volume of the mixture present in each well is 250 &#181;L.
	<ol>
		<li>To determine the required volume, the concentration of the viral stock should be known, and an MOI should be selected. For example, if the MOI is 2, then the number of viral particles added should be twice the number of cells. Calculate the volume V of the viral stock required using the formula: <img alt="Equation 5" src="/files/ftp_upload/64279/64279eq05.jpg" style="margin: auto;" />, with n being the number of viral particles needed, and C the known concentration of the viral stock</li>
	</ol>
	</li>
	<li>Incubate the contents of the 96-well plate for 5 days. After 5 days, transfer the contents of each well to a 1.5 mL tube.</li>
	<li>Centrifuge the tubes at 120 x g for 8 min to separate the cells from the virus-containing culture medium. Subject the cell pellets as well as the supernatants to DNA extraction followed by real-time PCR quantification to check for the presence of viral genomes in each fraction, thus assessing the infectivity of the viral particles.</li>
</ol>

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The authors declare no conflicts of interest.

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 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&#39;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 Sj&#246;gren&#39;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&#160;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.

<table><tbody><tr><td>0.2 mL thin-walled PCR tubes</td><td>Thermo Scientific</td><td>AB0620</td><td>Should be autoclaved before use</td></tr><tr><td>0.2-10 &amp;micro;L Microvolume Filter Tips</td><td>Corning</td><td>4807</td><td>Should be autoclaved before use</td></tr><tr><td>0.5-10 &amp;micro;L Pipette</td><td>BrandTech</td><td>704770</td><td></td></tr><tr><td>10 mL Disposable Serological Pipette</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>1000 &amp;micro;L Filtered Pipette Tips</td><td>QSP</td><td>TF-112-1000-Q</td><td></td></tr><tr><td>100-1000 &amp;micro;L Pipette</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>100x20 mm Cuture Plates</td><td>Sarstedt</td><td>83.1802</td><td></td></tr><tr><td>10-100 &amp;micro;L Pipette</td><td>BrandTech</td><td>704774</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>200 &amp;micro;L Filtered Pipette Tips</td><td>QSP</td><td>TF-108-200-Q</td><td></td></tr><tr><td>20-200 &amp;micro;L Pipette</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>50 mL Conical Tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>CFX96 Real-Time C-1000 Thermal Cycler</td><td>Bio-Rad</td><td>184-1000</td><td></td></tr><tr><td>DMSO</td><td>Amresco</td><td>0231</td><td></td></tr><tr><td>DNase/RNase Free Water</td><td>Zymo Research</td><td>W1001-1</td><td></td></tr><tr><td>EBER Primers</td><td>Macrogen</td><td>N/A</td><td>Custom Made Primers</td></tr><tr><td>EBV DNA Control (Standards)</td><td>Vircell</td><td>MBC065</td><td></td></tr><tr><td>Ethanol (Laboratory Reagent Grade)</td><td>Fischer Chemical</td><td>E/0600DF/17</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F9665</td><td></td></tr><tr><td>Fresco 21 MicroCentrifuge</td><td>Thermo Scientific</td><td>10651805</td><td></td></tr><tr><td>Glycogen Solution</td><td>Qiagen</td><td>158930</td><td></td></tr><tr><td>Hemocytometer</td><td>BOECO</td><td>BOE 01</td><td></td></tr><tr><td>Inverted Light Microscope</td><td>Zeiss</td><td>Axiovert 25</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>172-5121</td><td></td></tr><tr><td>Microcentrifuge Tube</td><td>Costar (Corning)</td><td>3621</td><td>Should be autoclaved before use</td></tr><tr><td>P3HR-1 Cell Line</td><td>ATCC</td><td>HTB-62</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>Phenol</td><td>VWR</td><td>20599.297</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>Pipette Filler</td><td>Thermo Scientific</td><td>9501</td><td></td></tr><tr><td>Precision Wipes</td><td>Kimtech</td><td>7552</td><td></td></tr><tr><td>RPMI-1640 Culture Medium</td><td>Sigma</td><td>R7388</td><td></td></tr><tr><td>SL 16R Centrifuge</td><td>Thermo Scientific</td><td>75004030</td><td></td></tr><tr><td>Sodium Acetate</td><td>Riedel-de Ha&amp;euml;n (Honeywell)</td><td>25022</td><td></td></tr><tr><td>Spectrophotomer</td><td>DeNovix</td><td>DS-11</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T-3253</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Water Jacketed CO2 Incubator</td><td>Thermo Scientific</td><td>4121</td><td></td></tr></tbody></table>

isolation and quantification of epstein-barr virus from the p3hr1 cell line

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&#39;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 &#176;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 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/&#181;L were optimal and resulted in high EBV concentrations. The concentration of viral particles obtained from the optimization protocol was 917,471 viral particles/&#181;L.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64279/64279fig02v2.jpg" /> 
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&#39;s t-test was performed comparing PMA/PMA and DMSO-treated cells to control cells; * indicates p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64279/64279fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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).

Watch this Scientific Journal Video about Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line at JoVE.com

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

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 ...

isolation-quantification-epstein-barr-virus-from-p3hr1-cell

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.6K Views.  American University of Beirut. Met deze methode kunnen we Epstein Barr-virusdeeltjes uit de P3HR1-cellijn isoleren en de virusvoorraad kwantificeren, zodat deze voor verschillende onderzoeksdoeleinden kan worden gebruikt. Het belangrijkste voordeel van deze techniek is dat het een relatief eenvoudige, goedkope, snelle en betrouwbare methode biedt om een EB-virale voorraad te bereiden. De vitale deeltjes die door deze techniek worden geproduceerd, kunnen in veel in vitro of in vivo experimentele modellen worden gebruikt voo...

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

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Biochemistry

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have specific sample requirements, high sample input, are low throughput, and are difficult to standardize, all of which are detrimental to characterization of cell therapy products during their development and manufacturing.
The assays described herein accurately identify and quantify immune cell types in a heterogeneous mixture of cells using isolated genomic DNA (gDNA). DNA methylation patterns are revealed through bisulfite conversion, a process in which unmethylated cytosines are converted to uracils. Unmethylated DNA regions are detected through qPCR amplification using primers targeting converted areas. One unique locus per assay is measured and serves as an accurate identifier for a specific cell type. The assays are robust and identify CD8+, regulatory, and Th17 T cells in a high throughput manner. These optimized assays can potentially be used for in-process and product release testing for cell therapy process.

Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells.

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

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

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