J.G.E.Y was the recipient of an Ontario Veterinary College PhD Scholarship and an Ontario Graduate Scholarship. This work was funded by Natural Sciences and Engineering Research Council of Canada Discovery Grants to SKW (grant #304737) and LS (grant #401127).

All work involving the use of animals was approved by the University of Guelph Animal Care Committee in accordance with the Canadian Council on Animal Care. All work is performed in a BioSafety Level 2 (BSL2) laboratory in Canada where mesogenic NDV is a Risk Group 2 Pathogen. All steps involved in the amplification and purification of NDV should be performed in a Type IIA biological safety cabinet for safety and sterility purposes.
1. Amplification of NDV using specified pathogen-free e...

<ol>
	<li>Kim, S. H., Samal, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+as+a+vaccine+vector+for+development+of+human+and+veterinary+vaccines.">Newcastle disease virus as a vaccine vector for development of human and veterinary vaccines.</a> Viruses. 8 (7), (2016).</li><li>Kortekaas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rift+Valley+fever+virus+immunity+provided+by+a+paramyxovirus+vaccine+vector.">Rift Valley fever virus immunity provided by a paramyxovirus vaccine vector.</a> Vaccine. 28 (27), 4394-4401 (2010).</li><li>Matveeva, O. V., Kochneva, G. V., Zainutdinov, S. S., Ilyinskaya, G. V., Chumakov, P. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+(NDV):+brief+history+of+its+oncolytic+strains.">Newcastle disease virus (NDV): brief history of its oncolytic strains.</a> Journal of Clinical Virology. 16 (1), 1-15 (2000).</li><li>Matuszewska, K., 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=Combining+vascular+normalization+with+an+oncolytic+virus+enhances+immunotherapy+in+a+preclinical+model+of+advanced-stage+ovarian+cancer.">Combining vascular normalization with an oncolytic virus enhances immunotherapy in a preclinical model of advanced-stage ovarian cancer.</a> Clinical Cancer Research. 25 (5), 1624-1638 (2019).</li><li>McAusland, T. 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=Combining+vanadyl+sulfate+with+Newcastle+disease+virus+potentiates+rapid+innate+immune-mediated+regression+with+curative+potential+in+murine+cancer+models.">Combining vanadyl sulfate with Newcastle disease virus potentiates rapid innate immune-mediated regression with curative potential in murine cancer models.</a> Molecular Therapy Oncolytics. 20, 306-324 (2021).</li><li>Warner, B. 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=Intranasal+vaccination+with+a+Newcastle+disease+virus-vectored+vaccine+protects+hamsters+from+SARS-CoV-2+infection+and+disease.">Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease.</a> iScience. 24 (11), 103219 (2021).</li><li>Sun, W., 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=Newcastle+disease+virus+(NDV)+expressing+the+spike+protein+of+SARS-CoV-2+as+a+live+virus+vaccine+candidate.">Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.</a> EBioMedicine. 62, (2020).</li><li>Sun, W., 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=A+Newcastle+disease+virus+(NDV)+expressing+a+membrane-anchored+spike+as+a+cost-effective+inactivated+SARS-CoV-2+vaccine.">A Newcastle disease virus (NDV) expressing a membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine.</a> Vaccines. 8 (4), 1-14 (2020).</li><li>Xu, Q., 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=Evaluation+of+Newcastle+disease+virus+mediated+dendritic+cell+activation+and+cross-priming+tumor-specific+immune+responses+ex+vivo.">Evaluation of Newcastle disease virus mediated dendritic cell activation and cross-priming tumor-specific immune responses ex vivo.</a> International Journal of Cancer. 146 (2), 531-541 (2020).</li><li>Burman, B., Pesci, G., Zamarin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+at+the+forefront+of+cancer+immunotherapy.">Newcastle disease virus at the forefront of cancer immunotherapy.</a> Cancers. 12 (12), 1-15 (2020).</li><li>Ricca, J. 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=Pre-existing+immunity+to+oncolytic+virus+potentiates+its+immunotherapeutic+efficacy.">Pre-existing immunity to oncolytic virus potentiates its immunotherapeutic efficacy.</a> Molecular Therapy. 26 (4), 1008-1019 (2018).</li><li>Zamarin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+oncolytic+virotherapy+overcomes+systemic+tumor+resistance+to+immune+checkpoint+blockade+immunotherapy.">Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy.</a> Science Translational Medicine. 6 (226), (2014).</li><li>Schirrmacher, V., van Gool, S., Stuecker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+therapy+resistance:+an+update+on+oncolytic+Newcastle+disease+virus+for+improvements+of+cancer+therapy.">Breaking therapy resistance: an update on oncolytic Newcastle disease virus for improvements of cancer therapy.</a> Biomedicines. 7 (3), (2019).</li><li>Santry, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+purification+of+high-titer+Newcastle+disease+virus+for+use+in+preclinical+mouse+models+of+cancer.">Production and purification of high-titer Newcastle disease virus for use in preclinical mouse models of cancer.</a> Molecular Therapy Methods and Clinical Development. 9, 181-191 (2018).</li><li>Cassel, W. A., Murray, D. 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Viruses used as therapeutic agents in preclinical studies must be highly purified to avoid toxicity when administered in vivo15. If adventitious agents or contaminants are not removed, this can lead to severe adverse reactions negating the therapeutic effect of the viral agent28. As NDV is produced in embryonated chicken eggs, there are several contaminating egg proteins, such as ovalbumin, that must be removed prior to its use in vivo in preclinical or cl...

Newcastle Disease Virus, also known as Avian Orthoavulavirus-1, is an enveloped avian paramyxovirus with the potential to be used both as an oncolytic virus or a viral-vectored vaccine1,2,3,4,5,6,7. Most recently, NDV engineered to express the spike protein of SARS-CoV-2 has been characterized as an effective intranasal vaccine in mouse and hamster challenge models7,8,9. When used as a cancer immunotherapy, it results in the recruitment of innate immune cells, specifically natural killer cells, production of type I interferon, and the generation of antitumor-specific T cells10,11,12,13. In addition to these potent immunostimulatory properties, NDV has a strong safety profile and a well-established reverse genetics system14,15. These desirable characteristics have prompted the evaluation of NDV in numerous preclinical and human clinical trials (NCT04871737, NCT01926028, NCT04764422)16,17. To further advance this highly promising, immune-stimulatory viral vector, detailed methods are needed for producing and purifying high-titer, ultra-pure NDV that can be safely administered in vivo.
As NDV is an avian paramyxovirus, it is most frequently amplified in embryonated chicken eggs. While there are cell-based systems available for propagating NDV, most have been unable to produce titers similar to that achieved in embryonated chicken eggs18. Nevertheless, there are some drawbacks to producing NDV in eggs, including the fact that egg-based production is lengthy and not easily scalable, sourcing large quantities of SPF chicken eggs can be problematic, and there exists the potential for contamination with egg allergens13,18,19,20. Recently, one group has shown that Vero cells grown in suspension in serum-free medium can support the replication of NDV to titers comparable to those achieved in eggs, prior to purification21. However, this required serial passaging of the virus to adapt the virus to Vero cells, and the optimization of a method to purify NDV from suspension Vero cells is still required21.
As highlighted previously, methods used for purifying high-titer, in vivo-grade virus vary depending on the virus in question22. There is a well-established reverse genetics system available for the generation of recombinant NDV. This process, involving the use of a cDNA clone, helper plasmids, and a helper virus expressing T7 RNA polymerase, has been previously described in detail15,23. This protocol can be applied to either lentogenic or mesogenic NDV. The virus described in this protocol is a recombinant mesogenic NDV encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria inserted between viral P and M genes as an individual transcription unit, as this has previously been described as the optimal site for foreign transgene insertion24.
Enclosed methods outline the purification of NDV based on its size, ranging from 100 to 500 nm, and its density15. This has allowed for the generation of in vivo-grade, high-titer NDV stocks in approximately 3 weeks, starting from when the eggs are received to having a final titer. Techniques frequently used in the large-scale production of egg-based viruses such as tangential flow filtration, depth filtration, and density gradient ultracentrifugation are described, enabling the translation of these methods to larger-scale production. Previously described techniques for the purification of NDV have been improved by the incorporation of a virus-stabilizing buffer, use of iodixanol during density gradient ultracentrifugation, and the description of various quality control measures to ensure in vivo-grade quality15. This has allowed for the purification of in vivo-grade NDV reaching titers as high as 3 &#215; 1010 PFU/mL from 0.8 to 1.0 L of allantoic fluid.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.02</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Slip-Tip Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Luer-Lok Syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Povidone Iodine Solution</td>
			<td>LORIS</td>
			<td>109-08</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Thermo-Fisher</td>
			<td>14955240</td>
			<td></td>
		</tr>
		<tr>
			<td>18G x 1 1/2 in Blunt Fill Needle</td>
			<td>BD</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>18G x 1 1/2 in Precision Glide Needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 5/8 in Needle</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo-Fisher</td>
			<td>03446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis Solution 37.5:1</td>
			<td>BioRad</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde-PBS</td>
			<td>Thermo-Fisher</td>
			<td>J19943-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Luer-Lok Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Tissue Culture Plate - Flat Bottom</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>Thermo-Fisher</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Froggabio</td>
			<td>A87-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488 Goat-Anti-Mouse</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-14 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B08861</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>BioRad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach (5%)</td>
			<td>Thermo-Fisher</td>
			<td>36-102-0599</td>
			<td></td>
		</tr>
		<tr>
			<td>Broad, unserrated tipped forceps</td>
			<td>Thermo-Fisher</td>
			<td>09-753-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>114405-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centramate Cassette Holder</td>
			<td>PALL</td>
			<td>CM018V</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+</td>
			<td>BioRad</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo-Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-259</td>
			<td>Thermo-Fisher</td>
			<td>BP101-50</td>
			<td></td>
		</tr>
		<tr>
			<td>DF1 Cells</td>
			<td>ATCC</td>
			<td>CRL-12203</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet Gel Recovery</td>
			<td>ClearH2O, INC</td>
			<td>72-01-1062</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital 1502 Sportsman Egg Incubator</td>
			<td>Berry Hill</td>
			<td>1502W</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Candler</td>
			<td>Berry Hill</td>
			<td>A46</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Thermo-Fisher</td>
			<td>BP82031GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA) solution, pH 8.0, 0.5 M in H2O</td>
			<td>Thermo-Fisher</td>
			<td>BP2482-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Threaded Tee fittings, nylon, 1/8 in NPT(F)</td>
			<td>Cole-Parmer</td>
			<td>06349-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Point High Precision Forceps</td>
			<td>Thermo-Fisher</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>ZEISS AXIO</td>
			<td></td>
			<td>Not necessary if not performing IFA or if NDV does not encode a fluorescent protein</td>
		</tr>
		<tr>
			<td>Freeze Dry System Freezone 4.5</td>
			<td>LABCONCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GiBOX Gel Imager</td>
			<td>Syngene</td>
			<td></td>
			<td>Imaging of Agarose Gels</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo-Fisher</td>
			<td>G33-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Thermo-Fisher</td>
			<td>BP381-5</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Thermo-Fisher</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>High Glucose Dulbecco&#39;s Modified Essential Medium</td>
			<td>Cytiva</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Kit</td>
			<td>Berry Hill</td>
			<td>3030</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>60% (w/v) solution of iodixanol in water (sterile)</td>
		</tr>
		<tr>
			<td>L-Lysine Monohydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>62929-100G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Male and Female Luer-Lok a 1/8 in national pipe thread, NPT</td>
			<td>Cole-Parmer</td>
			<td>41507-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive</td>
			<td>Cole-Parmer</td>
			<td>RK-07522-20</td>
			<td>Peristaltic Pump with digital display</td>
		</tr>
		<tr>
			<td>Masterflex L/S Easy Load Pump Head for Precision Tubing</td>
			<td>Cole-Parmer</td>
			<td>RK-07514-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Silicon tubing (Platinum) L/S 16</td>
			<td>Cole-Parmer</td>
			<td>96420-16</td>
			<td>BioPharm Platinum-Cured Silicone</td>
		</tr>
		<tr>
			<td>MC Pro 5 Thermocycler</td>
			<td>Eppendorf</td>
			<td>EP950040025</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Thermo-Fisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Protean Tetra Cell</td>
			<td>BioRad</td>
			<td>1658000EDU</td>
			<td>SDS-PAGE cast and running appartus</td>
		</tr>
		<tr>
			<td>Mouse-Anti-NDV</td>
			<td>Novus Biologicals</td>
			<td>NBP2-11633</td>
			<td>Clone 6H12</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>AB7481</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Thermo-Fisher</td>
			<td>85124</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Membrane LV Centramate Cassette, 100K</td>
			<td>PALL</td>
			<td>OS100T02</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>OWL Easycast B1A Mini Gel Electrophoresis System</td>
			<td>Thermo-Fisher</td>
			<td>B1A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10X Solution</td>
			<td>Thermo-Fisher</td>
			<td>BP399-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(Ethylene Glycol) Average Mn 20,000</td>
			<td>Sigma-Aldrich</td>
			<td>81300-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>PowePac 300</td>
			<td>BioRad</td>
			<td></td>
			<td>Model 1655050 - for Agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Q5 High Fidelity 2X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0492S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIA Amp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td></td>
		</tr>
		<tr>
			<td>RedSafe</td>
			<td>Thermo-Fisher</td>
			<td>50999562</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-a-lyzer Dialysis Cassette (Extra Strength), 10,000 MWCO 0.5-3 mL</td>
			<td>Thermo-Fisher</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Thermo-Fisher</td>
			<td>BP166-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (Pellets)</td>
			<td>Thermo-Fisher</td>
			<td>S318-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Specific pathogen free eggs</td>
			<td>CFIA</td>
			<td>NA</td>
			<td>Supplier will vary depending on location</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Thermo-Fisher</td>
			<td>S5-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Supracap 50 Depth Filter</td>
			<td>PALL</td>
			<td>SC050V100P</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Thermo-Fisher</td>
			<td>08-951-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sw41Ti Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td>Used in protocol step 2.3.1, 2.3.6, 2.3.7</td>
		</tr>
		<tr>
			<td>SX4750 Rotor</td>
			<td>Beckman Coulter</td>
			<td>369702</td>
			<td></td>
		</tr>
		<tr>
			<td>SxX4750 Adaptor for Concial-Bottom Tubes</td>
			<td>Beckman Coulter</td>
			<td>359472</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Invitrogen</td>
			<td>15524-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-Wall Ultraclear centrifuge tubes (9/16 in x 3 1/2 in)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo-Fisher</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Screw Clamp</td>
			<td>PALL</td>
			<td>88216</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Utility Pressure Gauges</td>
			<td>Cole-Parmer</td>
			<td>68355-06</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of high-titer recombinant newcastle disease virus from allantoic fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Harvesting allantoic fluid 
As allantoic fluid is harvested from embryonated chicken eggs, it should appear clear and transparent. If the fluid appears opaque and yellow, this indicates the presence of contaminants. Inclusion of this allantoic fluid during purification will impede the purification process, as the pressure will quickly rise and surpass 10 psi, resulting in the shearing of the virus and loss of infectious virus. Allantoic fluid that appears bloody suggests that the eggs were inoculate...

Watch this Scientific Journal Video about Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid at JoVE.com

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

This protocol outlines the techniques used to produce Newcastle Disease Virus so that it can be administered safely to mice and other animals such as hamsters and sheep. The use of tangential flow filtration allows for large starting volumes to be processed more efficiently compared to existing procedures that use only centrifugation processes. This is a lengthy procedure involving several intricate steps, meaning time management may be an issue.I would recommend identifying all potential pause steps so that the technique can be split up appropriately, minimizing stress. Demonstrating this procedure will be Alex, a research assistant from my laboratory. For the purification of Newcastle Disease Virus or NDV, first, prime the previously set experimental system by running 50 milliliters of PBS through it.Stop the pump when approximately five milliliters of PBS are left in the tube. Next, attach a depth filter with a one to three micromolar retention rating to the tubing. To vent the filter, remove the second cap on the epical side of the depth filter then start running an additional 50 milliliters of PBS through the system.Once the PBS begins to flow through the vent at the top of the filter, close the port and continue to flow PBS through the lines. After that replace the waste vessel with a new sterile collection vessel and begin to run Allantoic Fluid through the depth filter. Next, set up the tangential flow filtration or TFF system in an open confirmation and run as described in the manuscript.When there are about five milliliters of fluid left in the reservoir, pause the pump and add depth filtered Allantoic Fluid to the reservoir. Then resume the pump flow. To prevent shearing of the virus, it's important to ensure that the pressure of the system does not exceed 10 pound force per square inch.To increase the illusion, a combination of the speed of the peristaltic pump and C-clamp should be used. 150 to 100 milliliters of Allantoic Fluid are left in the reservoir, pause the pump and exchange the buffer by adding 150 to 200 milliliters of ML buffer. Again, pause the pump when five to 10 milliliters of fluid are left in the reservoir, then using two C-clamps close the waistline.Uncouple the retented line feeding the reservoir and insert it into a 50 milliliter conical tube. Then resume the flow and pause when there are few drops of fluid left in the reservoir tank. Next, remove the C-clamps from the waste lines and reattach the retented feed line to the reservoir tank.Meanwhile, to prepare for iodixanol density gradient ultracentrifugation, first add a 0.5 milliliters of 40%iodixanol to a 13.2 milliliter open top thin wall ultra centrifuge tube. Then make the column ready with successive additions of 2.5 milliliters of 20%iodixanol, and 2.5 milliliters of 10%iodixanol. Before ultracentrifugation, carefully add six to 6.5 milliliters of the virus solution alluded from the TFF system to the iodixanol column without disturbing the gradient.After ultracentrifugation, remove the tubes with a pair of sterile forceps. The target band should be a large band between the 10 and 20%gradients. Next, suspend the tube over the top of a beaker using a retort stand.After that, attach a 1.5 inch needle of 18 gauge to a five milliliter syringe then puncture the side of the ultracentrifuge tube with the needle and remove the target band by slowly extending the plunger. After removing iodixanol from the virus solution as described in the manuscript, use a syringe to carefully inject the virus into a dialysis cassette. To concentrate the virus solution, remove the dialysis cassette from the dialysis buffer and place it in a small sealable plastic bag.Then add a 15 to 25 milliliters of 40%polyethylene glycol to the bag so that the dialysis cassette is completely submerged. After dialysis briefly rinse the dialysis cassette with PBS. Next, fill a syringe fitted with a 1.5 inch needle of 18 gauge with air, insert the syringe into the dialysis cassette and push the plunger.Rotate the apparatus for removing the virus without removing any of the introduced air. To dislodge residual virus adhering to the membrane, carefully massage the membrane with gloved fingers without damaging it. To ease the dislodging process, remove some of the excess air in the dialysis cassette.For quality control assays, first, dilute the virus solution 100 times by adding 10 microliters of it to 990 microliters of PBS in a 1.5 milliliter tube. Then continue serial dilution by adding 100 microliters of the previous dilution to 900 microliters of PBS. For infection assays, add 20 microliters of each delusion to each well of a 96 well plate.Before examining the cytopathic effect of green fluorescence protein, or performing an indirect immunofluorescence assay, incubate the plate at 37 degrees Celsius with 5%carbon dioxide for at least 48 hours. By using this protocol, highly purified NDV was obtained as demonstrated by sodium do sulfate polyacrylamide gel electrophoresis of the viral proteins. The infectivity of the virus purified by this method was also verified by the 50%tissue culture infection dose or TCID50 assay done in 96 well plates.The TCID50 was determined from the number of positive wells for each dilution by using the Spearman-Karber calculator. The infection assay revealed the difference between the cytopathic effects of lentogenic and mesogenic NDV on DF one cells under different conditions after 10 and 24 hours of incubation. The immuno florence assay was also effective in studying the infection of Vero cells by lentogenic NDV.When priming the experimental system, ensure that the lines are primed effectively as any residual sodium hydroxide will inactivate the virus. Also is important to maintain membrane integrity as compromising this will significantly impair virus purification and yield.

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JoVE Journal

Cancer Research

3.3K visualizações.  University of Guelph. Este protocolo descreve as técnicas usadas para produzir o Newcastle Disease Virus para que possa ser administrado com segurança para camundongos e outros animais, como hamsters e ovelhas. O uso da filtragem de fluxo tangencial permite que grandes volumes iniciais sejam processados de forma mais eficiente em comparação com os procedimentos existentes que utilizam apenas processos de centrifugação. Este é um procedimento demorado envolvendo várias etapas complexas, o que significa que ...