Name: growth, purification, and titration of oncolytic herpes simplex virus
Uploaded: 2021-05-13T14:30:00
Description: Watch this Scientific Journal Video about Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus at JoVE.com

Research in the Saha lab was supported in part by funds from the DOD (W81XWH-20-1-0702) and Dodge Jones Foundation-Abilene. Samuel D. Rabkin and Melissa R.M. Humphrey were partially supported by NIH (R01 CA160762).

1) oHSV growth
NOTE: Ensure institutional biosafety committee approval before working with oHSV. This study was conducted under approved IBC Protocol no. 18007. Maintain BSL2 precautions: bleach all pipets, tips, tubes, and other materials that come into contact with the virus. Spray gloves with 70% isopropyl alcohol before hands leave the BSL2 cell culture hood. Always thoroughly wash hands with soap water after working with a virus.
<ol>
	<li>On day -1, seed low-passage Vero cells in 20 T-...

<ol>
	<li>Harrington, K., Freeman, D. J., Kelly, B., Harper, J., Soria, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+oncolytic+virotherapy+in+cancer+treatment.">Optimizing oncolytic virotherapy in cancer treatment.</a> Nature Reviews Drug Discovery. 18 (9), 689-706 (2019).</li><li>Zhang, S., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+and+development+of+oncolytic+viruses:+are+they+the+future+of+cancer+immunotherapy.">The discovery and development of oncolytic viruses: are they the future of cancer immunotherapy.</a> Expert Opinion on Drug Discovery. 16 (4), 391-410 (2021).</li><li>Bommareddy, P. K., Peters, C., Saha, D., Rabkin, S. D., Kaufman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+herpes+simplex+viruses+as+a+paradigm+for+the+treatment+of+cancer.">Oncolytic herpes simplex viruses as a paradigm for the treatment of cancer.</a> Annual Review of Cancer Biology. 2 (1), 155-173 (2018).</li><li>Peters, C., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+herpes+viruses+as+oncolytics.">Designing herpes viruses as oncolytics.</a> Molecular Therapy-Oncolytics. 2, 15010 (2015).</li><li>Nguyen, H. -. M., Saha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+oncolytic+herpes+simplex+virus+for+glioblastoma+treatment.">The current state of oncolytic herpes simplex virus for glioblastoma treatment.</a> Oncolytic Virotherapy. 10, 1-27 (2021).</li><li>Koch, M. S., Lawler, S. E., Chiocca, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+oncolytic+viruses+from+bench+to+bedside:+an+overview+of+current+clinical+trials.">HSV-1 oncolytic viruses from bench to bedside: an overview of current clinical trials.</a> Cancers. 12 (12), 3514 (2020).</li><li>Menotti, L., Avitabile, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+oncolytic+immunovirotherapy:+the+blossoming+branch+of+multimodal+therapy.">Herpes simplex virus oncolytic immunovirotherapy: the blossoming branch of multimodal therapy.</a> International Journal of Molecular Sciences. 21 (21), 8310 (2020).</li><li>Nguyen, H. M., Guz-Montgomery, K., Saha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virus+encoding+a+master+pro-inflammatory+cytokine+interleukin+12+in+cancer+immunotherapy.">Oncolytic virus encoding a master pro-inflammatory cytokine interleukin 12 in cancer immunotherapy.</a> Cells. 9 (2), 400 (2020).</li><li>Agarwalla, P. K., Aghi, M. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+growth,+preparation,+and+assay.">Herpes simplex virus growth, preparation, and assay.</a> Methods in Molecular Biology. 2060, 57-72 (2020).</li><li>Froechlich, G., 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=Integrity+of+the+antiviral+STING-mediated+DNA+sensing+in+tumor+cells+is+required+to+sustain+the+immunotherapeutic+efficacy+of+herpes+simplex+oncolytic+virus.">Integrity of the antiviral STING-mediated DNA sensing in tumor cells is required to sustain the immunotherapeutic efficacy of herpes simplex oncolytic virus.</a> Cancers. 12 (11), 3407 (2020).</li><li>Froechlich, G., 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=Generation+of+a+novel+mesothelin-targeted+oncolytic+herpes+virus+and+implemented+strategies+for+manufacturing.">Generation of a novel mesothelin-targeted oncolytic herpes virus and implemented strategies for manufacturing.</a> International Journal of Molecular Sciences. 22 (2), 477 (2021).</li><li>Mosca, J. D., Pitha, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+posttranscriptional+regulation+of+exogenous+human+beta+interferon+gene+in+simian+cells+defective+in+interferon+synthesis.">Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.</a> Molecular and Cellular Biology. 6 (6), 2279-2283 (1986).</li><li>Gousseinoz, E., Kools, W., Pattnaik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+impurity+reduction+in+viral+vaccine+manufacturing.">Nucleic acid impurity reduction in viral vaccine manufacturing.</a> BioProcess International. 12 (2), 59-68 (2014).</li><li>Diefenbach, R. J., Fraefel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+methods+and+protocols.">Herpes simplex virus: methods and protocols.</a> Methods in Molecular Biology. , (2014).</li><li>Hadi, A. M., 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+experimental+trial+to+prepared+&#947;1+34.5+herpes+simplex+virus+1+immunogene+by+cloning+technique.">An experimental trial to prepared &#947;1 34.5 herpes simplex virus 1 immunogene by cloning technique.</a> Systematic Review Pharmacy. 11 (5), 140-149 (2020).</li><li>Kuroda, T., Martuza, R. L., Todo, T., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flip-Flop+HSV-BAC:+bacterial+artificial+chromosome+based+system+for+rapid+generation+of+recombinant+herpes+simplex+virus+vectors+using+two+independent+site-specific+recombinases.">Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases.</a> BMC Biotechnology. 6, 40 (2006).</li><li>Motamedifar, M., Noorafshan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytopathic+effect+of+the+herpes+simplex+virus+type+1+appears+stereologically+as+early+as+4+h+after+infection+of+Vero+cells.">Cytopathic effect of the herpes simplex virus type 1 appears stereologically as early as 4 h after infection of Vero cells.</a> Micron. 39 (8), 1331-1334 (2008).</li><li>Blaho, J. A., Morton, E. R., Yedowitz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+propagation,+quantification,+and+storage.">Herpes simplex virus: propagation, quantification, and storage.</a> Current Protocols in Microbiology. , 1 (2005).</li><li>Malenovska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+stabilizers+and+rates+of+freezing+on+preserving+of+structurally+different+animal+viruses+during+lyophilization+and+subsequent+storage.">The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage.</a> Journal of Applied Microbiology. 117 (6), 1810-1819 (2014).</li><li>Vahlne, A. G., Blomberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+herpes+simplex+virus.">Purification of herpes simplex virus.</a> Journal of General Virology. 22 (2), 297-302 (1974).</li><li>Sathananthan, B., Rodahl, E., Flatmark, T., Langeland, N., Haarr, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+herpes+simplex+virus+type+1+by+density+gradient+centrifugation+and+estimation+of+the+sedimentation+coefficient+of+the+virion.">Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion.</a> APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 105 (3), 238-246 (1997).</li><li>Mundle, S. 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The protocol starts with the growth of oHSV in low-passage Vero cells. The confluency of the Vero cell monolayer should be ~80% at the time of virus inoculation as overgrown cells can develop tight fibrous structures that can reduce oHSV entry into Vero cells20. Once 90-100% CPE is observed, the culture supernatant is removed, cells are harvested, resuspended in VB/supernatant (see step 1.4.6), snap-frozen, and stored at -80 &#176;C&#160;for later purification. Blaho and colleagues employed a slig...

Oncolytic viruses (OVs) are an emerging and unique form of cancer immunotherapy. OVs selectively replicate in and lyse tumor cells (sparing normal/healthy cells)1 while inducing anti-tumor immunity2. Oncolytic herpes simplex virus (oHSV) is one of the most extensively studied viruses among all OVs. It is furthest along in the clinic, with Talimogene laherparepvec (T-VEC) being the first and only OV to receive FDA approval in the USA for the treatment of advanced melanoma3. In addition to T-VEC, many other genetically engineered oHSVs are being tested preclinically and clinically in different cancer types3,4,5,6,7,8. The current advanced recombinant DNA biotechnology has further increased the feasibility of engineering new oHSVs coding for therapeutic transgene(s)3,5. An efficient system of oHSV propagation, purification, and titer determination is critical before any (newly developed) oHSV can be tested for in vitro and in vivo studies. This paper describes a simple step-by-step method of oHSV growth (in Vero cells), purification (by the sucrose-gradient method), and titration (by an oHSV plaque assay in Vero cells) (Figure 1). It can be easily adopted in any Biosafety Level 2 (BSL2) laboratory setting to achieve a high-quality viral stock for preclinical studies.
Vero, an African green monkey kidney cell line, is the most commonly used cell line for oHSV propagation9,10,11,12,13 as Vero cells have a defective antiviral interferon signaling pathway14. Other cell lines with inactivated stimulator of interferon genes (STING) signaling can also be used for oHSV growth12,13. This protocol utilizes Vero cells for oHSV growth and&#160;plaque assay. Following propagation, oHSV-infected cells are harvested, lysed, and subjected to purification, wherein lysed cells are first treated with benzonase nuclease to degrade host cell DNA, prevent nucleic acid-protein aggregation, and reduce the viscosity of the cell lysate. As proper activation of benzonase often requires Mg2+, 1-2 mM MgCl2 is used in this protocol15. The host cell debris from the benzonase-treated cell lysate is further eliminated by serial filtration before high-speed sucrose-gradient centrifugation. A viscous 25% sucrose solution cushion helps to ensure a slower rate of virus migration through the sucrose layer, leaving host cell-related components in the supernatant, thus improving purification and limiting virus loss in the pellet16. The purified oHSV is then titrated on Vero cells, and viral plaques are visualized by Giemsa staining17 or X-gal staining (for LacZ encoding oHSVs)18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL centrifuge tubes</td>
			<td>Sigma</td>
			<td>CLS3620</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma</td>
			<td>E8263-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Corning</td>
			<td>MT-10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>MT-21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH3007003</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Sigma</td>
			<td>G3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-262-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>MT-21-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Glucose Dulbecco&#8217;s Phosphate-buffered Saline</td>
			<td>Sigma</td>
			<td>D4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human immune globulin</td>
			<td>Gamastan</td>
			<td>NDC 13533-335-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 250 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 500 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25C</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red solution</td>
			<td>Sigma</td>
			<td>N4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher scientific</td>
			<td>&#160;15710S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate rocker</td>
			<td>Fisher</td>
			<td>88861043</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferricyanide</td>
			<td>Sigma</td>
			<td>P8131</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferrocyanide</td>
			<td>Sigma</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75004381</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 21R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002446</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-371</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.45 PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.8 MCE</td>
			<td>MilliporeSigma</td>
			<td>SLAA033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 5 &#181;m PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>T150 culture flask</td>
			<td>Falcon</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>MP Biomedicals LLC</td>
			<td>816116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic water bath</td>
			<td>Branson</td>
			<td>CPX-952-116R</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Corning</td>
			<td>46-101-RF</td>
			<td></td>
		</tr>
	</tbody>
</table>

growth, purification, and titration of oncolytic herpes simplex virus

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a &#34;plaque assay&#34; in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.

A brief overview of the entire protocol is depicted in Figure 1, which represents the critical steps involved in the growth, purification, and titration of oHSV. CPE in Vero cells can be detected as early as 4 h post-HSV infection19. Figure 2 demonstrates CPE in Vero cells at three different time points following oHSV infection. The level of the CPE is increased over time. In this protocol, 90-100% CPE is usually observed within 48 h of l...

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Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use.

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

Oncolytic herpes simplex virus is an emergent form of cancer immunotherapy. An efficient system of virus propagation, purification, and titeric determination is critical for the use in experimental studies. This method is very simple and can be easily adopted in any biosafety level two laboratory setting to receive a high quality viral stock for preclinical studies.Production of a high quality, pure viral stock is crucial since it is used to study anticancer immune response and to develop a novel form of virus based cancer immunotherapy. This protocol can be used to purify any wild-type or genetically engineered herpes simplex virus. This protocol should be easy to read and easy to follow for an individual who have never performed this technique before.The listed materials and reagents should be in place before trying this technique for the first time. Begin by washing the T-150 square centimeter flasks with two times high glucose DPBS supplemented with 1%IFCS. Aspirate the DPBS and add seven milliliters of virus inoculum per flask.Gently rock the flasks for five minutes and incubate at 37 degrees Celsius for 1.5 to 2 hours. Then, remove the inoculum and add 25 milliliters of DMEM supplemented with 1%IFCS to each flask. After incubation at 37 degrees Celsius and 5%carbon dioxide for two to four days, collect 20 milliliters of the culture supernatant from each flask into 50 milliliter centrifuge tubes.Using a cell scraper, scrape the cells from the bottom of the flasks. Add approximately 15 milliliters of the previously collected culture supernatant to each flask to bring the volume up to 20 milliliters, then use a 10 milliliter sterile serological pipette to gently wash the bottom of the flasks a few times. Collect the cells with medium into 50 milliliter conical centrifuge tubes placed on ice.Centrifuge the tubes at 300 times G for 10 minutes at four degrees Celsius. Resuspend each pellet thoroughly in 1.25 milliliters of virus buffer, or VB, and 1.25 milliliters of previously collected culture supernatant. Mix all resuspended pellets into one 50 milliliter conical centrifuge tube.Snap freeze the resuspended cells using dry ice and 100%ethanol and store at minus 80 degrees Celsius. Thaw the previously snapped frozen cells in a warm water bath at 37 degrees Celsius and sonicate for one minute for three cycles in a water bath. Add 175 units of Benzonase Nuclease and two microliters of two millimolar magnesium chloride per milliliter of the cell lysate and vortex.After an incubation of 30 minutes and warm water bath at 37 degrees Celsius, place the tube on ice. Spin the cell lysate at 300 times G for 10 minutes. Collect the supernatant in a new 50 milliliter conical centrifuge tube and resuspend the cell pellet in 500 microliters of VB.Designate it as pellet one.Spin the collected supernatant again at 500 times G for 10 minutes and store the supernatant separately in a fresh 50 milliliter conical centrifuge tube to use later. Resuspend the cell pellet in 500 microliters of VB, designating it as pellet two. Combine the resuspended pellet one and pellet two in a new 1.75 milliliter centrifuge tube and vortex and sonicate twice in a water bath before spinning the tubes at 400 times G for 10 minutes.Collect the supernatant from 1.7 milliliter centrifuge tube and combine it with the supernatant previously collected 50 milliliter conical centrifuge tube. Filter the supernatant through a sterile five micrometer PVDF membrane filter into a new 50 milliliter centrifuge tube called tube one. Add one milliliter of VB to the 50 milliliter centrifuge tube, emptied after filtering the supernatant from the previous step, vortex, and pass it through the same PVDF membrane filter placed on tube one.Pass the filtrate from tube one through a sterile 0.8 micrometer MCE membrane filter placed on a new 50 milliliter centrifuge tube called tube two. Add one milliliter of VB to the emptied tube one, vortex, and pass it through the same MCE filter placed on tube two. Pass the filtrate from tube two through a sterile 0.45 micrometer PVDF filter placed on a new 50 milliliter centrifuge tube labeled tube three.As described before, repeat the washing of tube two with one milliliter of VB to add filtrate to tube three. In this representative analysis, the cytopathic effect could be observed in Vero cells at 36, 48, and 72 hours post oHSV infection with the rounding of the affected cells. The increasing level of CPE over time is evident as visualized by mCherry expression.At 72 hours post infection, viral plaques were stained with X-gal to visualize oHSVs with lacZ expression. Gently scrubbing off virus infected cells and gently washing the bottom on the flask is critical to keep all HSV infected cell intact so that a high titer viral stock can be obtained.

growth-purification-and-titration-of-oncolytic-herpes-simplex-virus

Research

JoVE Journal

Immunology and Infection

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 ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

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 ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...