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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JoVE Journal

Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

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 ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Isolation and Genome Analysis of Single Virions using &#039;Single Virus Genomics&#039;

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...