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. K. <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+virus+engineering+and+preparation.">Oncolytic herpes simplex virus engineering and preparation.</a> Methods in Molecular Biology. 797, 1-19 (2012).</li><li>Grosche, L., 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=Herpes+simplex+virus+type+1+propagation,+titration+and+single-step+growth+curves.">Herpes simplex virus type 1 propagation, titration and single-step growth curves.</a> Bio-protocol. 9 (23), 3441 (2019).</li><li>Sutter, S. O., Marconi, P., Meier, A. 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. 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=High-purity+preparation+of+HSV-2+vaccine+candidate+ACAM529+is+immunogenic+and+efficacious+in+vivo.">High-purity preparation of HSV-2 vaccine candidate ACAM529 is immunogenic and efficacious in vivo.</a> PLoS One. 8 (2), 57224 (2013).</li><li>Jiang, 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=Immobilized+cobalt+affinity+chromatography+provides+a+novel,+efficient+method+for+herpes+simplex+virus+type+1+gene+vector+purification.">Immobilized cobalt affinity chromatography provides a novel, efficient method for herpes simplex virus type 1 gene vector purification.</a> Journal of Virology. 78 (17), 8994-9006 (2004).</li><li>Grosche, L., 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=Herpes+simplex+virus+type+1+propagation,+titration+and+single-step+growth+curves.">Herpes simplex virus type 1 propagation, titration and single-step growth curves.</a> Bio-protocol. 9 (23), 3441 (2019).</li><li>Svennerholm, B., 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=Separation+of+herpes+simplex+virus+virions+and+nucleocapsids+on+Percoll+gradients.">Separation of herpes simplex virus virions and nucleocapsids on Percoll gradients.</a> Journal of Virological Methods. 1 (6), 303-309 (1980).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> Journal of Visualized Experiments: JoVE. (93), e52065 (2014).</li><li>Miyatake, S., Iyer, A., Martuza, R. L., 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=Transcriptional+targeting+of+herpes+simplex+virus+for+cell-specific+replication.">Transcriptional targeting of herpes simplex virus for cell-specific replication.</a> Journal of Virology. 71 (7), 5124-5132 (1997).</li><li>Fabiani, M., Limongi, D., Palamara, A. T., De Chiara, G., Marcocci, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+titrate+herpes+simplex+virus-1+(HSV-1)+using+laser-based+scanning+of+near-infrared+fluorophores+conjugated+antibodies.">A novel method to titrate herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies.</a> Frontiers in Microbiology. 8, 1085 (2017).</li></ol>

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

6.1K visualizações.  Texas Tech University Health Sciences Center. O vírus herpes simplex oncolítico é uma forma emergente de imunoterapia contra o câncer. Um sistema eficiente de propagação, purificação e determinação titeric do vírus é fundamental para o uso em estudos experimentais. Este método é muito simples e pode ser facilmente adotado em qualquer configuração de laboratório de nível dois de biossegurança para receber um estoque viral de alta qualidade para estudos pré-clínicos.A produção de um estoque viral puro e de alt...