Name: isolation and quantification of epstein-barr virus from the p3hr1 cell line
Uploaded: 2022-09-28T13:10:00
Description: Watch this Scientific Journal Video about Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line at JoVE.com

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 ...

<ol>
	<li>Epstein, M. A., Achong, B. G., Barr, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+particles+in+cultured+lymphoblasts+from+Burkitt's+lymphoma.">Virus particles in cultured lymphoblasts from Burkitt's lymphoma.</a> Lancet. 1 (7335), 702-703 (1964).</li><li>Sample, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+types+1+and+2+differ+in+their+EBNA-3A,+EBNA-3B,+and+EBNA-3C+genes.">Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.</a> Journal of Virology. 64 (9), 4084-4092 (1990).</li><li>Chang, M. S., Kim, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+in+human+malignancy:+a+special+reference+to+Epstein-Barr+virus+associated+gastric+carcinoma.">Epstein-Barr virus in human malignancy: a special reference to Epstein-Barr virus associated gastric carcinoma.</a> Cancer Research and Treatment. 37 (5), 257-267 (2005).</li><li>Manet, E., Schwab, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cancer. , 1602-1607 (2017).</li><li>Babcock, G. J., Decker, L. L., Volk, M., Thorley-Lawson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EBV+persistence+in+memory+B+cells+in+vivo.">EBV persistence in memory B cells in vivo.</a> Immunity. 9 (3), 395-404 (1998).</li><li>Khan, G., Miyashita, E. M., Yang, B., Babcock, G. J., Thorley-Lawson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+EBV+persistence+in+vivo+a+model+for+B+cell+homeostasis.">Is EBV persistence in vivo a model for B cell homeostasis.</a> Immunity. 5 (2), 173-179 (1996).</li><li>Jha, H. C., Banerjee, S., Robertson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+gammaherpesviruses+in+cancer+pathogenesis.">The role of gammaherpesviruses in cancer pathogenesis.</a> Pathogens. 5 (1), 18 (2016).</li><li>El-Sharkawy, A., Al Zaidan, L., Malki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-associated+malignancies:+roles+of+viral+oncoproteins+in+carcinogenesis.">Epstein-Barr virus-associated malignancies: roles of viral oncoproteins in carcinogenesis.</a> Frontiers in Oncology. 8, 265 (2018).</li><li>Vereide, D., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+evolution+of+lymphomas+induced+by+Epstein-Barr+virus.">Insights into the evolution of lymphomas induced by Epstein-Barr virus.</a> Advances in Cancer Research. 108, 1-19 (2010).</li><li>Vereide, D. T., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphomas+differ+in+their+dependence+on+Epstein-Barr+virus.">Lymphomas differ in their dependence on Epstein-Barr virus.</a> Blood. 117 (6), 1977-1985 (2011).</li><li>Houen, G., Trier, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+and+systemic+autoimmune+diseases.">Epstein-Barr virus and systemic autoimmune diseases.</a> Frontiers in Immunology. 11, 587380 (2020).</li><li>Ortiz, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Epstein-Barr+virus+infection+on+inflammatory+bowel+disease+(IBD)+clinical+outcomes.">Impact of Epstein-Barr virus infection on inflammatory bowel disease (IBD) clinical outcomes.</a> Revista Espanola de Enfermedades Digestivas. 114 (5), 259-265 (2021).</li><li>Caplazi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+rheumatoid+arthritis.">Mouse models of rheumatoid arthritis.</a> Veterinary Pathology. 52 (5), 819-826 (2015).</li><li>Kiesler, P., Fuss, I. J., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+inflammatory+bowel+diseases.">Experimental models of inflammatory bowel diseases.</a> Cellular and Molecular Gastroenterology and Hepatology. 1 (2), 154-170 (2015).</li><li>Warde, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+arthritis:+EBV+induces+arthritis+in+mice.">Experimental arthritis: EBV induces arthritis in mice.</a> Nature Reviews Rheumatology. 7 (12), 683 (2011).</li><li>Jog, N. R., James, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein+Barr+virus+and+autoimmune+responses+in+systemic+lupus+erythematosus.">Epstein Barr virus and autoimmune responses in systemic lupus erythematosus.</a> Frontiers in Immunology. 11, 623944 (2020).</li><li>Shimizu, N., Yoshiyama, H., Takada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+propagation+of+Epstein-Barr+virus+(EBV)+recombinants+in+EBV-negative+Akata+cells.">Clonal propagation of Epstein-Barr virus (EBV) recombinants in EBV-negative Akata cells.</a> Journal of Virology. 70 (10), 7260-7263 (1996).</li><li>Hsu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Epstein-Barr+virus+(EBV)+reactivation+in+Raji+cells+by+doxorubicin+and+cisplatin.">Induction of Epstein-Barr virus (EBV) reactivation in Raji cells by doxorubicin and cisplatin.</a> Anticancer Research. 22, 4065-4071 (2002).</li><li>Nutter, L. M., Grill, S. P., Li, J. S., Tan, R. S., Cheng, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+virus+enzymes+by+phorbol+esters+and+n-butyrate+in+Epstein-Barr+virus+genome-carrying+Raji+cells.">Induction of virus enzymes by phorbol esters and n-butyrate in Epstein-Barr virus genome-carrying Raji cells.</a> Cancer Research. 47 (16), 4407-4412 (1987).</li><li>Fresen, K. O., Cho, M. S., zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+transforming+EBV+from+non-producer+cells+after+superinfection+with+non-transforming+P3HR-1+EBV.">Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.</a> International Journal of Cancer. 22 (4), 378-383 (1978).</li><li>Glaser, R., Tarr, K. L., Dangel, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transforming+prototype+of+Epstein-Barr+virus+(B95-8)+is+also+a+lytic+virus.">The transforming prototype of Epstein-Barr virus (B95-8) is also a lytic virus.</a> International Journal of Cancer. 44 (1), 95-100 (1989).</li><li>Sairenji, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Epstein-Barr+virus+(EBV)+release+from+P3HR-1+and+B95-8+cell+lines+by+monoclonal+antibodies+to+EBV+membrane+antigen+gp350/220.">Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.</a> Journal of Virology. 62 (8), 2614-2621 (1988).</li><li>Savage, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assessment+of+the+population+of+cotton-top+tamarins+(Saguinus+oedipus)+and+their+habitat+in+Colombia.">An assessment of the population of cotton-top tamarins (Saguinus oedipus) and their habitat in Colombia.</a> PLoS one. 11 (12), 0168324 (2016).</li><li>Kallin, B., Klein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+carried+by+raji+cells:+a+mutant+in+early+functions.">Epstein-Barr virus carried by raji cells: a mutant in early functions.</a> Intervirology. 19 (1), 47-51 (1983).</li><li>Fresen, K. O., Cho, M. S., Hausen, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+transforming+EBV+from+non-producer+cells+after+superinfection+with+non-transforming+P3HR-1+EBV.">Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.</a> International Journal of Cancer. 22 (4), 378-383 (1978).</li><li>Bounaadja, L., Piret, J., Goyette, N., Boivin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Epstein-Barr+virus,+human+herpesvirus+6+(HHV-6),+and+HHV-8+antiviral+drug+susceptibilities+by+use+of+real-time-PCR-based+assays.">Evaluation of Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-8 antiviral drug susceptibilities by use of real-time-PCR-based assays.</a> Journal of Clinical Microbiology. 51 (4), 1244-1246 (2013).</li><li>Buelow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+four+real-time+PCR+methods+for+the+quantitative+detection+of+Epstein-Barr+virus+from+whole+blood+specimens.">Comparative evaluation of four real-time PCR methods for the quantitative detection of Epstein-Barr virus from whole blood specimens.</a> Journal of Molecular Diagnostics. 18 (4), 527-534 (2016).</li><li>Wu, D. Y., Kalpana, G. V., Goff, S. P., Schubach, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus+nuclear+protein+2+(EBNA2)+binds+to+a+component+of+the+human+SNF-SWI+complex,+hSNF5/Ini1.">Epstein-Barr virus nuclear protein 2 (EBNA2) binds to a component of the human SNF-SWI complex, hSNF5/Ini1.</a> Journal of Virology. 70 (9), 6020-6028 (1996).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EBNA2-deleted+Epstein-Barr+virus+(EBV)+isolate,+P3HR1,+causes+Hodgkin-like+lymphomas+and+diffuse+large+B+cell+lymphomas+with+type+II+and+Wp-restricted+latency+types+in+humanized+mice.">EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice.</a> PLoS Pathogens. 16 (6), 1008590 (2020).</li></ol>

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 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;L Microvolume Filter Tips</td>
			<td>Corning</td>
			<td>4807</td>
			<td>Should be autoclaved before use</td>
		</tr>
		<tr>
			<td>0.5-10 &#181;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 &#181;L Filtered Pipette Tips</td>
			<td>QSP</td>
			<td>TF-112-1000-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000 &#181;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 &#181;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 &#181;L Filtered Pipette Tips</td>
			<td>QSP</td>
			<td>TF-108-200-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;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&#235;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...

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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 ...

This method will allow us to isolate Epstein Barr virus particles from the P3HR1 cell line and quantify the virus stock so that it can be used for various research purposes. The main advantage of this technique is that it provides a relatively easy, inexpensive, fast, and reliable method to prepare an EB viral stock. The vital particles produced by this technique can be used in many in vitro or in vivo experimental models for the diagnosis of EB.To begin, prepare a volume of cell suspension A in a 15 milliliter conical tube, adjust the volume such that the number of cells is roughly 2.2 million cells for a 100 millimeter culture plate.Add five milliliters of complete culture medium to the tube. Add 80 microliters of dimethyl sulfoxide to the tube. Then add 350 microliters of one milligram per milliliter PMA to the tube.The final concentration of PMA should be 35 nanograms per milliliter, and that of DMSO should be 0.08%Add 4.27 milliliters of culture medium so that the total volume is 10 milliliters. Mix the contents of the tube by tilting, then transfer the contents to a 100 millimeter culture plate. Leave the plate in a cell culture incubator for five days at 37 degrees Celsius, 5%carbon dioxide.After five days in the incubator centrifuge the contents of the plate at 120 G for eight minutes to pellet the cells Collect the cell-free virus containing supernatant and discard the cell pellet. Again centrifuge the supernatant at 16, 000 G for 90 minutes at four degree Celsius to pellet the virus particles. Discard the supernatant and resuspend the virus pellet in five milliliters of culture medium, Aliquot the viral suspension into 20 tubes each containing 250 microliters of the viral suspension, and store the suspension at minus 80 degrees Celsius.To separate the proteins from DNA add 500 microliters of Tris-saturated phenol to one of the tubes containing the viral preparation. Add 100 microliters of water and vortex well to obtain a pink emulsion. Centrifuge for 15 minutes at 9, 650 G, collect the supernatant and transfer it to a new 1.5 milliliter micro centrifuge tube.Add an equivalent of one-tenth of the supernatant volume of cold sodium acetate and mix by pipetting up and down. Then add one microliter of 20 milligrams per milliliter glycogen, and mix by pipetting. Add three times the supernatant's volume of cold 100%ethanol and store it at minus 80 degrees Celsius overnight.To isolate the viral DNA centrifuge at 9, 650 G for 15 minutes at four degrees Celsius. Discard the supernatant to obtain a DNA pellet and wash the pellet three times with one milliliter of cold 70%ethanol. Again centrifuge at 9, 650 G for 15 minutes and discard the supernatant.After air drying the pellet for about 10 minutes, resuspend the pellet in 10 to 50 microliters of nuclease free distilled water. Store the samples at minus 20 degrees Celsius for later processing or at four degrees Celsius overnight to ensure maximal DNA dissolution, followed by storage at minus 20 degrees Celsius. After cleaning the pedestal of a micro spectrophotometer with a delicate task wiper load one microliter of the prepared DNA sample and note the concentration of the DNA in the sample.Check the ratios of absorbance at both 260 to 280 nanometers, and 260 to 230 nanometers. DNA will absorb at 260 nanometers, proteins at 280 nanometers, and organic substances such as phenol will absorb at 230 nanometers. A ratio of 1.8 to 2 is considered sufficient for realtime PCR.To prepare the PCR reaction mixes, place five microliters of a cyber green realtime PCR mix in 0.2 milliliter PCR tubes. Add one microliter of 7.5 pico moles per microliter forward primer and one microliter of 7.5 pico moles per microliter of reverse primer to each tube. Here, EBV small RNA 2 gene is amplified.To determine the EBV genome copy number. Then add two microliters of nuclease free water and one microliter of viral DNA to one of the tubes. The total volume of the reaction mixture is 10 microliters.Prepare other tubes that will be used as standards with known EBV genome copy numbers. Run the PCR mixtures starting with an initial step of activation at 95 degrees Celsius for five minutes. Then 40 cycles at 95 degrees Celsius for 15 seconds, and at 58 degrees Celsius for 30 seconds.Export all data sheets as CSV files and calculate the log of the number of EBV genome copies per standard tube and mean CQ values. Generate a qPCR standard curve by plotting the CT values of the standards against the log of the number of genome copies. Employing the standard curve plot equation, derive the number of EBV genome copies in the PCR tube containing the induced viral DNA.Finally, use the formula shown on the screen to calculate the concentration of the induced viral preparation. Here X is the number of EBV genome copies derived from the standard curve, and F is the dilution factor used to set up the DNA per PCR reaction. Cell counting using a hemocytometer chamber is shown here.Four quadrants are counted using a light microscope. Here 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. An optimization trial was performed to determine the concentrations of PMA and dimethyl sulfoxide that would yield the highest number of EBV particles.A DMSO concentration of 0.8%and a PMA concentration of 35 nanograms per microliters were optimal and resulted in high EBV concentrations. Our lab used the vital particles produced by this technique to examine pro autoimmune or inflammatory responses to this virus. As well as to develop novel anti-EBV agents.

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