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-150 cm2 flasks at a density of 7-8 &#215; 106 cells/flask in regular Vero cell medium. 
	NOTE: Vero medium is prepared by supplementing Dulbecco&#39;s modified eagle medium (DMEM) with 10% heat-inactivated fetal calf serum (IFCS).</li>
	<li>On Day 0 (cells are 80-90% confluent), add oHSV inoculum on the Vero cells.
	<ol>
		<li>Preparation of virus inoculum
		<ol>
			<li>Use a multiplicity of infection (MOI) of 0.01 (MOI can vary from 0.01 to 0.1 depending on the replication capacity of virus) for virus amplification. Use formula (1) to calculate the amount of virus (mL) for 20 flasks. 
			&#8203;Amount of virus (mL) for 20 flasks = amount of virus needed (pfu)/titer of virus stock (pfu/mL)(1)</li>
			<li>Use high-glucose Dulbecco&#39;s phosphate-buffered saline (DPBS) supplemented with 1% IFCS to prepare the virus inoculum (7 mL per T-150 cm2 flask, i.e., ~140 mL for 20 flasks). Add the required amount of oHSV to 140 mL of high-glucose DPBS/1% IFCS solution, vortex for 1 min, and keep the mixture ready for addition to Vero cells.</li>
		</ol>
		</li>
		<li>Wash the T-150 cm2 flasks 2x with high-glucose DPBS supplemented with 1% IFCS (10 mL/wash). Aspirate the DPBS/1% IFCS and add 7 mL of virus inoculum/T-150 cm2 flask.</li>
		<li>Gently rock the flasks for 5 min using a flask rocker for proper distribution of the inoculum over the Vero monolayer, and then incubate the flasks at 37 &#176;C for 1.5-2 h. Make sure the incubator shelf is level.</li>
		<li>Remove the inoculum and add DMEM supplemented with 1% IFCS (25 mL/flask). Incubate the flasks for 2-4 days.</li>
	</ol>
	</li>
	<li>Check the flasks daily for 90-100% CPE (see Figure 2).</li>
	<li>Harvest the oHSV-infected Vero cells.
	<ol>
		<li>Collect the culture supernatant (~20 mL) from each flask (leave ~5 mL in each flask) in 50 mL conical centrifuge tubes or media container.</li>
		<li>Use a cell scraper to scrape the cells from the bottom of the flasks gently. 
		NOTE: The cells should quickly come off the flasks.</li>
		<li>Add ~15 mL of the culture supernatant (collected in step 1.4.1) to each flask (which brings the volume of ~20 mL in each flask, i.e., 400 mL for 20 flasks) and gently wash the bottom of the flasks a few times using a 10 mL sterile serological pipet. 
		NOTE: Do not pipet vigorously. Aim to keep all cells intact.</li>
		<li>Collect the cells (+ medium) into 50 mL conical centrifuge tubes on ice (use 8 tubes to hold 400 mL of harvested cells from 20 flasks).</li>
		<li>Spin the cells at 300 g for 10 min at 4 &#176;C and aspirate the supernatant.</li>
		<li>Add 1.25 mL (50%) of Virus Buffer (VB) and 1.25 mL (50%) of culture supernatant (collected in step 1.4.1) to each centrifuge tube and re-suspend each pellet thoroughly. Transfer the re-suspended cells from eight 50 mL centrifuge tubes to one 50 mL conical centrifuge tube. 
		NOTE: Refer to the preparation of VB solution in Table 1. Filter-sterilize the VB solution using a media sterilization filter. Use 0.5 mL of VB + 0.5 mL of supernatant per T-150 cm2 flask for re-suspension, i.e., for 20 flasks (20 mL), use 10 mL of VB and 10 mL of culture supernatant.</li>
		<li>Snap-freeze the re-suspended cells using dry ice/100% ethanol and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2) oHSV purification
<ol>
	<li>Snap-freeze (in dry ice and 100% ethanol)/thaw (in a 37 &#176;C warm water bath) the cells followed by water bath-sonication for 1 min for a total of 3 cycles to ensure proper lysis of the cells to release virus in the supernatant. 
	NOTE: Perform sonication for 1 min using 40 kHz, 120 V power. If a tunable sonicator is not available, use an ultrasonic water bath sonicator. Take a 50 &#956;L aliquot for titration in section 3, which will help to identify which of the following step(s) is responsible for potential virus loss during the purification procedure.</li>
	<li>Treat the cell lysate with Benzonase Nuclease (175 units/mL) + 2 mM MgCl2 (1 M stock = 2 &#181;L/mL), vortex, and incubate for 30 min at 37 &#176;C. Place the tube on ice and perform the following steps at 4 &#176;C.</li>
	<li>Pellet the cell debris by low-speed centrifugation.
	<ol>
		<li>Spin the cell lysate at 300 g for 10 min.</li>
		<li>Collect the supernatant in a new 50 mL conical centrifuge tube (re-suspend the cell pellet in 0.5 mL of VB, designate it as pellet-1, and store at 4 &#176;C for use in step 2.3.5; see Figure 1).</li>
		<li>Spin the supernatant (obtained in step 2.3.2) again at 500 g for 10 min.</li>
		<li>Collect the supernatant in a new 50 mL conical centrifuge tube (re-suspend the cell pellet in 0.5 mL of VB, designate it as pellet-2, and store at 4 &#176;C for use in step 2.3.5; see Figure 1).</li>
		<li>Combine the re-suspended pellet-1 (from 2.3.2) and pellet-2 (from 2.3.4) into a new 1.7 mL centrifuge tube, vortex/sonicate (water bath) 2x, and spin at 400 g for 10 min. Collect the supernatant and combine it with the supernatant obtained in step 2.3.4; see Figure 1). 
		NOTE: Perform sonication for 1 min using 40 kHz, 120 V power to prevent viral aggregation before the filtration procedure in step 2.4. Take a 50 &#956;L aliquot of the combined supernatant for titration in section 3.</li>
	</ol>
	</li>
	<li>Filter the combined supernatant (~21 mL, i.e., 20 mL from 1.4.6, 0.5 mL from 2.3.2, and 0.5 mL from 2.3.4) using the following 3-step filtration method.
	<ol>
		<li>Draw 21 mL of the supernatant using a 10 mL syringe (5-7 mL each time for easy passage through the filter) and pass it through a sterile 5 &#956;m polyvinylidene difluoride (PVDF) membrane filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 1).
		<ol>
			<li>Add 1 mL of VB to a 50 mL conical centrifuge tube emptied in 2.4.1 (to collect the remaining trace amount of virus supernatant), vortex, and pass through same 5 &#956;m PVDF filter placed on TUBE 1 (bringing the total to 22 mL of filtrate). Proceed to step 2.4.2.</li>
		</ol>
		</li>
		<li>Draw 22 mL of the filtrate from TUBE 1 (as in 2.4.1) and pass it through a sterile 0.8 &#956;m mixed cellulose ester (MCE) membrane filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 2).
		<ol>
			<li>Add 1 mL of VB to TUBE 1 emptied in step 2.4.2, vortex, and pass it through the same 0.8 &#956;m MCE filter placed on TUBE 2 (bringing the total to 23 mL of filtrate). Proceed to step 2.4.3.</li>
		</ol>
		</li>
		<li>Draw 23 mL of filtrate from TUBE 2 (as in 2.4.1) and pass it through a sterile 0.45 &#181;m PVDF filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 3).
		<ol>
			<li>Add 1 mL of VB to TUBE 2 emptied in step 2.4.3, vortex, and pass it through the same 0.45 &#956;m PVDF filter placed on TUBE 3 (bringing the total to 24 mL of filtrate). 
			NOTE: Take a 50 &#956;L aliquot of the filtrate for titration in section 3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>High-speed centrifugation using the sucrose-gradient method 
	NOTE: A fixed angle F13-14x50cy rotor was used in this protocol. Both the rotor and the centrifuge must be at 4 &#176;C before proceeding with the following steps.
	<ol>
		<li>Add 10 mL of an ice-cold, sterile-filtered 25% sucrose solution (prepared by dissolving 25 g of sucrose powder in 100 mL of Hank&#39;s Balanced Salt Solution) in a new 50 mL conical centrifuge tube.</li>
		<li>Slowly (3 mL/min) add 24 mL of the virus filtrate (obtained from step 2.4.3.1) on top of the sucrose layer. Take care to maintain separate layers of the virus filtrate and the sucrose solution. 
		NOTE: Up to 30 mL of the virus layer can be added over 10 mL of the sucrose solution.</li>
		<li>Centrifuge the tube for 90 min at 22,620 g at 4 &#176;C.</li>
		<li>Remove the supernatant and the sucrose layer from the 50 mL conical centrifuge tube. 
		NOTE: The virus pellet should be whitish. Take a 50 &#956;L aliquot of the supernatant and a 50 &#956;L aliquot of the sucrose layer for titration in section 3.</li>
	</ol>
	</li>
	<li>Re-suspend the pellet in 10% glycerol/PBS solution.
	<ol>
		<li>Add 1 mL of sterile 10% glycerol (diluted in PBS) to the 50 mL conical centrifuge tube to cover the pellet. 
		NOTE: The amount of the re-suspended mixture can vary (such as 0.8-1.2 mL) depending on the pellet size. A smaller volume would give a higher concentration of virus.</li>
		<li>Place the 50 mL conical centrifuge tube on ice for 2-4 h. During this period, sonicate/vortex the pellet every 15 min for 30 s to help dislodge/re-suspend the pellet.</li>
		<li>Collect the re-suspended pellet (1 mL of 10% glycerol/PBS + the pellet size will bring the total volume to ~1.3 mL) in a 2 mL microcentrifuge tube. [Optional: Add another 0.3 mL of 10% glycerol/PBS to the same 50 mL conical centrifuge tube to collect the remaining trace amount of the pellet, pipet up and down, and combine with the re-suspended pellet in step 2.6.3 to bring the total volume to ~1.6 mL.]
		<ol>
			<li>If the suspension in step 2.6.3 is turbid or cloudy (due to cell debris), centrifuge the tube at 500 g for 10 min, transfer the supernatant to a new 2 mL microcentrifuge tube, and proceed to step 2.6.4.</li>
		</ol>
		</li>
		<li>Take a 50 &#956;L aliquot for oHSV titration in section 3. Aliquot the rest of the solution (250 &#956;L/aliquot) into sterile microcentrifuge tubes with screw caps (sealed well for long-term storage), snap-freeze, and store at -80 &#176;C until use (ready for experimental studies once the oHSV titer is determined). 
		&#8203;NOTE: All materials that come in contact with the virus must be bleached or treated with ultraviolet radiation before removal from the hood or disposal.</li>
	</ol>
	</li>
</ol>
3. oHSV titration and plaque assay
<ol>
	<li>Seed 1.7-1.8 x 105 Vero&#160;cells/well in a 6-well cell culture plate in Vero cell medium (DMEM with 10% IFCS; see step 1.1). 
	NOTE: Make sure that the cells are homogeneously distributed throughout the well; do not swirl the plate, which can cause accumulation of cells in the middle of the wells. To prevent swirling, slowly rock the plate by hand vertically, then horizontally, and then gently place the plate in the incubator.</li>
	<li>The next day (when the cells reach 70-80% confluency), aspirate the culture medium, and add 1 mL/well of high-glucose PBS supplemented with 1% IFCS. Leave the plate in the cell culture hood until step 3.3 is complete.</li>
	<li>Serially dilute the virus in 5 mL polypropylene tubes using PBS/1% IFCS (Figure 3). 
	NOTE: Use the 50 &#956;L aliquot collected in step 2.6.4 for serial dilution. In the 1st tube (10-3 dilution), add 2 &#956;L of the virus in 1998 &#956;L of PBS/1%IFCS, vortex. In the 2nd tube (10-5 dilution), take 10 &#956;L from the 1st tube in 990 &#956;L of PBS/1% IFCS, vortex. In the 3rd tube (10-6 dilution), take 100 &#956;L from the 2nd tube in 900 &#956;L of PBS/1% IFCS, vortex; continue this 10-fold serial dilution until 10-9 dilution. See the details in Figure 3.</li>
	<li>Aspirate PBS/1% IFCS, and add 0.7 mL/well of the serially diluted virus (starting from 10-5 to 10-9) to Vero cells. 
	NOTE: Do not let the cells dry.</li>
	<li>Gently rock the plate on a rocker for 5 min at room temperature (to ensure homogeneous distribution of the virus inoculum).</li>
	<li>Incubate the plate for 1.5 h at 37 &#176;C. 
	NOTE: During this incubation period, prepare 1:1000 dilution of human immunoglobulin G (IgG) in DMEM supplemented with 1% IFCS. For a 6-well plate, prepare 12.5 mL so that 2 mL/well can be used in step 3.7. Adjust the dilution to account for lot variations in the human IgG.</li>
	<li>Remove the virus inoculum from the wells, and add 2 mL/well of 0.1% human IgG (to neutralize the oHSV in the culture medium and prevent the formation of secondary plaques) in DMEM supplemented with 1% IFCS. Incubate the plate at 37 &#176;C for 3-4 days. 
	NOTE: Clear plaques usually form in 3 days.</li>
	<li>Fixing and staining plates
	<ol>
		<li>Remove the supernatant, and fix the cells in pure methanol (1 mL/well) for 5 min. Remove the methanol, and allow the plates to air-dry.</li>
		<li>Dilute Giemsa stain (1:5) with deionized water, and add 1 mL of the diluted Giemsa stain per well. Incubate the plate at room temperature for 10-15 min.</li>
		<li>Remove the stain, rinse with tap water, and allow the plates to air-dry.</li>
		<li>Count the plaques using a dissecting microscope.</li>
		<li>Optional for oHSVs with lacZ expression:
		<ol>
			<li>Remove the supernatant, and fix the cells with cold 0.2% glutaraldehyde/2% paraformaldehyde for 5-10 min at room temperature.</li>
			<li>Remove the fixative solution, and wash the cells 3x with PBS.</li>
			<li>Add X-gal solution to the cells (1 mL/well), and incubate the plate at 37 &#176;C for 2 h. 
			NOTE: The X-gal stain should be prepared freshly on the day of staining; long-term storage might lead to fading of color. See the preparation of X-gal solution in Table 1.</li>
			<li>Remove the X-gal stain, and wash the plate with tap water for 1 min.</li>
			<li>Counter-stain with Neutral Red solution (1 mL/well) for 2 min at room temperature. 
			NOTE: See preparation of Neutral Red solution in Table 1.</li>
			<li>Wash the plate with tap water for 1 min; allow the plates to air-dry.</li>
			<li>Count blue plaques using a dissecting microscope (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate the titer by using formula (2) 
	Titer in pfu/mL (plaque-forming units) = Number of plaques/0.7 mL &#215; dilution factor (2) 
	NOTE: For example, if 25 plaques are found in the 10-9 dilution well, the titer is 25/0.7 &#215; 109 = 35.7 &#215; 109 = 3.57 &#215; 1010 pfu/mL. This is the final oHSV titer of aliquots prepared in step 2.6.4.</li>
</ol>

<ol><li>Harrington, K., Freeman, D. J., Kelly, B., Harper, J., Soria, J. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+oncolytic+virotherapy+in+cancer+treatment%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">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/pubmed?term=((The+discovery+and+development+of+oncolytic+viruses%3A+are+they+the+future+of+cancer+immunotherapy%5BTitle%5D)+AND+%22Expert+Opinion+on+Drug+Discovery%22%5BJournal%5D)">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/pubmed?term=((Oncolytic+herpes+simplex+viruses+as+a+paradigm+for+the+treatment+of+cancer%5BTitle%5D)+AND+%22Annual+Review+of+Cancer+Biology%22%5BJournal%5D)">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/pubmed?term=((Designing+herpes+viruses+as+oncolytics%5BTitle%5D)+AND+%22Molecular+Therapy-Oncolytics%22%5BJournal%5D)">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/pubmed?term=((The+current+state+of+oncolytic+herpes+simplex+virus+for+glioblastoma+treatment%5BTitle%5D)+AND+%22Oncolytic+Virotherapy%22%5BJournal%5D)">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/pubmed?term=((HSV-1+oncolytic+viruses+from+bench+to+bedside%3A+an+overview+of+current+clinical+trials%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">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/pubmed?term=((Herpes+simplex+virus+oncolytic+immunovirotherapy%3A+the+blossoming+branch+of+multimodal+therapy%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((Oncolytic+virus+encoding+a+master+pro-inflammatory+cytokine+interleukin+12+in+cancer+immunotherapy%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Oncolytic+herpes+simplex+virus+engineering+and+preparation%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Herpes+simplex+virus+type+1+propagation%2C+titration+and+single-step+growth+curves%5BTitle%5D)+AND+%22Bio-protocol%22%5BJournal%5D)">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/pubmed?term=((Herpes+simplex+virus+growth%2C+preparation%2C+and+assay%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Integrity+of+the+antiviral+STING-mediated+DNA+sensing+in+tumor+cells+is+required+to+sustain+the+immunotherapeutic+efficacy+of+herpes+simplex+oncolytic+virus%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">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/pubmed?term=((Generation+of+a+novel+mesothelin-targeted+oncolytic+herpes+virus+and+implemented+strategies+for+manufacturing%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((Transcriptional+and+posttranscriptional+regulation+of+exogenous+human+beta+interferon+gene+in+simian+cells+defective+in+interferon+synthesis%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">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/pubmed?term=((Nucleic+acid+impurity+reduction+in+viral+vaccine+manufacturing%5BTitle%5D)+AND+%22BioProcess+International%22%5BJournal%5D)">Nucleic acid impurity reduction in viral vaccine manufacturing.</a> BioProcess International. 12 (2), 59-68 (2014).</li>
<li>Herpes simplex virus: methods and protocols. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. Diefenbach, R. J., Fraefel, C. , Humana Press. New York, USA. (2014).</li>
<li>Hadi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+experimental+trial+to+prepared+%CE%B31+34.5+herpes+simplex+virus+1+immunogene+by+cloning+technique%5BTitle%5D)+AND+%22Systematic+Review+Pharmacy%22%5BJournal%5D)">An experimental trial to prepared γ1 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/pubmed?term=((Flip-Flop+HSV-BAC%3A+bacterial+artificial+chromosome+based+system+for+rapid+generation+of+recombinant+herpes+simplex+virus+vectors+using+two+independent+site-specific+recombinases%5BTitle%5D)+AND+%22BMC+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Cytopathic+effect+of+the+herpes+simplex+virus+type+1+appears+stereologically+as+early+as+4+h+after+infection+of+Vero+cells%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">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/pubmed?term=((Herpes+simplex+virus%3A+propagation%2C+quantification%2C+and+storage%5BTitle%5D)+AND+%22Current+Protocols+in+Microbiology%22%5BJournal%5D)">Herpes simplex virus: propagation, quantification, and storage.</a> Current Protocols in Microbiology. , Chapter 14, Unit 14E 1(2005).</li>
<li>Malenovska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+stabilizers+and+rates+of+freezing+on+preserving+of+structurally+different+animal+viruses+during+lyophilization+and+subsequent+storage%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">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/pubmed?term=((Purification+of+herpes+simplex+virus%5BTitle%5D)+AND+%22Journal+of+General+Virology%22%5BJournal%5D)">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/pubmed?term=((Purification+of+herpes+simplex+virus+type+1+by+density+gradient+centrifugation+and+estimation+of+the+sedimentation+coefficient+of+the+virion%5BTitle%5D)+AND+%22APMIS%3A+Acta+Pathologica%2C+Microbiologica%2C+et+Immunologica+Scandinavica%22%5BJournal%5D)">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/pubmed?term=((High-purity+preparation+of+HSV-2+vaccine+candidate+ACAM529+is+immunogenic+and+efficacious+in+vivo%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Immobilized+cobalt+affinity+chromatography+provides+a+novel%2C+efficient+method+for+herpes+simplex+virus+type+1+gene+vector+purification%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">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/pubmed?term=((Herpes+simplex+virus+type+1+propagation%2C+titration+and+single-step+growth+curves%5BTitle%5D)+AND+%22Bio-protocol%22%5BJournal%5D)">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/pubmed?term=((Separation+of+herpes+simplex+virus+virions+and+nucleocapsids+on+Percoll+gradients%5BTitle%5D)+AND+%22Journal+of+Virological+Methods%22%5BJournal%5D)">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/pubmed?term=((Viral+concentration+determination+through+plaque+assays%3A+using+traditional+and+novel+overlay+systems%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((Transcriptional+targeting+of+herpes+simplex+virus+for+cell-specific+replication%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">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/pubmed?term=((A+novel+method+to+titrate+herpes+simplex+virus-1+%28HSV-1%29+using+laser-based+scanning+of+near-infrared+fluorophores+conjugated+antibodies%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">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>

SDR is a co-inventor on patents relating to oncolytic herpes simplex viruses, owned and managed by Georgetown University and Massachusetts General Hospital, which have received royalties from Amgen and ActiVec Inc. and is on the Scientific Advisory Board of EG 427. The other authors have nothing to disclose.

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><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&amp;rsquo;s Modified Eagle Medium</td><td>Corning</td><td>MT-10-013-CV</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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&#039;s Balanced Salt Solution (HBSS)</td><td>Corning</td><td>MT-21-021-CV</td><td></td></tr><tr><td>High-Glucose Dulbecco&amp;rsquo;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>&amp;nbsp;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 &amp;micro;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...

Watch this Scientific Journal Video about Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus at JoVE.com

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 ...

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

Research

JoVE Journal

Immunology and Infection

6.5K Views.  Texas Tech University Health Sciences Center. In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use. 

Video: Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Medicine

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Cancer Research

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Genetics

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

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 ...

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Summary

Explore More Videos

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

Production and Purification of Baculovirus for Gene Therapy Application

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Preparation of Viral DNA from Nucleocapsids

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

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

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Plaquing of Herpes Simplex Viruses