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).

<ol>
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The authors have no conflicts of interest to declare.

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 clinical models29. Even after intensive purification, ovalbumin is still found29. The removal of these contaminating proteins is a rigorous process requiring significant time and resources15,22. If NDV could be produced in a cell-based system at sufficiently high titers required for in vivo applications, this may simplify the purification process and improve the translation of NDV into&#160;clinical settings. However, egg-based production of oncolytic NDV has been used in numerous human clinical trials30.
At each step of the purification process, samples can be streaked on blood agar plates as an additional quality control measure to ensure the absence of bacterial contamination.
The pressure during depth filtration should not build up quickly. This is a sign of protein contamination and will significantly slow the purification process and require the use of additional depth filters. Depth filtration should not be completed rapidly; this step typically takes 1-1.5 h. Usually, rapid completion of the depth filtration step results in a lower viral titer.
As the virus is concentrated, a &#34;cloudy stream&#34; should be emerging into the reservoir from the retentate line. This suggests that there is virus in the allantoic fluid that is being concentrated as it passes through the cassette.
During both depth filtration and tangential flow filtration steps, the pressure should never exceed 10 psi. Exceeding this pressure will shear the virus. The addition of ML buffer to the allantoic fluid acts as a stabilizing agent and is hypothesized to prevent viral aggregation, resulting in the increased recovery of infectious virus without compromising filterability.
The above method outlines a suitable procedure to produce both lentogenic and mesogenic strains of NDV under BSL-2 conditions, since mesogenic NDV is not a select agent in Canada. This results in the generation of high-titer stocks suitable for use in animal&#160;models. Similar to other protocols in the literature that use size-exclusion purification methods, such as depth filtration and TFF, these methods limit the use of harsher, ultracentrifugation techniques where virus recovery is reduced compared to size-exclusion purification methods15.
The use of TFF provides an element of scalability as TFF can be used to concentrate large starting volumes, in contrast to ultracentrifugation techniques. Existing NDV purification techniques are improved upon in the above protocol through the substitution of sucrose for iodixanol in the ultracentrifugation step, which greatly reduces the chance of losing virus due to cassette rupture at the end of the dialysis step, and the addition of ML buffer, which improves filtration and increases yield.
The use of iodixanol gradients is preferred over sucrose gradients as iodixanol is less viscous and non-toxic to cells and provides a &#34;cleaner&#34; final product31,32. Iodixanol significantly reduced the levels of proinflammatory cytokines and chemokines present after density gradient ultracentrifugation of purified virus33. To our knowledge, this is the first time iodixanol has been used in the purification of NDV. The concentration of iodixanol in a solution can be determined by quantifying the absorbance at 340 nm with reference to a standard curve. This characteristic was used to determine that NDV bands at approximately 15% iodixanol and to confirm that it can be removed by dialysis (Supplemental Figure S1B). Iodixanol was initially developed as a contrast imaging agent, and we observed no adverse events when a 15% iodixanol solution was administered intravenously and intranasally to C57BL/6 mice. This suggests that dialysis to remove iodixanol may not be required, shortening the purification process by approximately 16 h. Additionally, sucrose is hygroscopic, causing the dialysis cassette to swell significantly, limiting the volume that can be injected and increasing the risk of cassette rupture and loss of virus34,35. When equal volumes of iodixanol- and sucrose-prepared virus were injected into dialysis cassettes, significantly less swelling occured in the iodixanol-prepared virus (compare Figure 4C and Supplemental Figure S3A). However, sucrose is a more suitable stabilization agent for NDV than iodixanol. This led to the supplementation of the iodixanol gradient with ML buffer. Prior to the addition of ML buffer to the purification process, there was a more intense band in the 10% iodixanol gradient observed in addition to the target band at 15% (Supplemental Figure S3B). This band likely contains damaged or incomplete virions generated during the purification process. Addition of ML buffer allows for better filtration of NDV while minimizing the hygroscopic nature of the density gradient medium, reducing the risk of rupturing the dialysis cassette. The swelling of the dialysis cassette can be further reduced by dialyzing in 1x PBS supplemented with 5% sucrose, as ultimately this is what the virus will be stored in.
As NDV is a negative sense, single-stranded RNA virus, isolating RNA and generating cDNA using random primers and specific primer sets for PCR allows for the sequencing of various regions of the genome from one cDNA reaction (Table 1). This is important as it allows the pathotype and integrity of the transgene and its transcriptional regulatory elements to be confirmed. The cleavage site of the lentogenic NDV F protein should be monobasic rather than polybasic, as NDV possessing polybasic cleavage sites are typically mesogenic or velogenic pathotypes, and classified as select agents36,37. This protocol also describes a reliable RT-qPCR method for the high-throughput quantification of NDV genomes. Prior to its use in animal models and following the completion of the other quality control assays, acute toxicity should be assessed in 8-week-old BALB/C mice. This strain is characterized as being more sensitive to virus-induced toxicities than other strains of mice15. Additionally, this will give some indication of the accuracy of the virus titer. For example, weight loss will be observed, but weight should begin to increase after 36-48 h. If this is not the case, then it is possible that the virus titer is higher than initially reported, which may be due to aggregation of viral particles. This initial result following the purification of a recombinant NDV led to the incorporation of the ML buffer throughout the purification process. This buffer has been suggested to improve the filterability and recovery of the virus during the purification processes38.
When quantifying the concentration of infectious virus by TCID50 the use of IFA may be necessary to visualize NDV infection, especially at higher dilutions when CPE may not be obvious, or if the virus does not express a fluorescent reporter gene (Figure 8). The primary antibody used for IFA is specific to the ribonucleoprotein of NDV, a protein complex that associates with the RNA genome during genome replication. As NDV is an avian virus, the chicken embryo fibroblast cell line, DF1, is an optimal cell line for assessing virus titer. Typically, virus-negative allantoic fluid is used as a means to provide the trypsin-like proteases required by lentogenic NDVs for F protein cleavage39. The use of virus-negative allantoic fluid can be avoided by supplementing with 125 &#181;g/mL trypsin while reducing FBS concentration to 2%. This provides a simple, cost-effective alternative to virus-negative allantoic fluid while still providing the trypsin-like proteases required. When comparing these two culture conditions at an MOI of 0.5 over a 24 h timespan, there appears to be little difference between the two, with the medium supplemented with trypsin potentially resulting in more CPE (Figure 7). Other cell lines such as Vero cells can be used with the same culture conditions; however, the CPE is less pronounced. Using the above protocol with DF1 cells, NDV CPE should be evident within 24 h. Specifically, one should see the formation of syncytia by 24 h (Figure 7E-H). Using the described purification procedure, we consistently purified NDV to titers ranging from 1 &#215; 109-3 &#215; 1010 PFU/mL from a starting volume of 0.8-1.0 L of allantoic fluid.
Overall, the described protocol improves upon previously published NDV purification procedures by using iodixanol as a density gradient medium instead of sucrose and by the addition of ML buffer to improve filtration. In addition, we describe a complementary method for titering NDV, include a detailed RT-qPCR method for determining NDV copy number, and define optimal conditions for the use of trypsin instead of allantoic fluid during virus titration. Although this process generates high-titer NDV stocks that can be administered systemically at high doses, this procedure involves multiple steps, which can increase variability among users and introduce the potential for contamination. An additional limitation is the requirement for the starting material to be of high quality. It is critical that the allantoic fluid be free of contaminating yolk as this greatly reduces the efficiency of the filtration and size-exclusion steps. Overall, this process is time-consuming but can be shortened by omitting the dialysis step. This procedure results in a greater yield of in vivo-grade virus compared to other purification methods15,18. The ability to produce higher-quality, more concentrated NDV extends its capabilities to be used as an oncolytic agent or vaccine vector in animal models of disease in both preclinical and clinical settings.

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><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>CO&lt;sub&gt;2 &lt;/sub&gt;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&#039;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 inoculated with too many PFU of virus. The yield from this batch of eggs will be significantly lower than expected and it is unlikely that this virus will be able to be used in vivo. Eggs inoculated with lentogenic NDV should still have vasculature evident after 72 h and the embryos should not have died, as visualized in Figure 1. If they have, this suggests that embryo has been damaged or contaminated in some way.
Iodixanol density gradient ultracentrifugation 
Following ultracentrifugation, a high-titer, white, NDV-containing band should be located between the 10% and 20% gradients (Figure 4B).
Coomassie stain of SDS-PAGE gels 
Figure 5 demonstrates the difference between an unpurified (Lane 2) and purified virus stock (Lane 3). A comparison of the two shows a significant decrease in the intensity of contaminating protein bands and the emergence of dominant NDV bands in the purified virus stock.
Quantification of viral titer by TCID50 
If using the recommended conditions when titering a concentrated stock of NDV, CPE should be evident after 24 h (Figure 6). In comparison to a lentogenic strain NDV of similar titer, the CPE is more pronounced in the mesogenic strain at the same time point.
Acute toxicity analysis in 8-week-old BALB/C mice 
When 1 &#215; 108 PFU were administered intravenously, mice should be monitored for adverse effects such as weight loss &#62;20%, ruffled coat, hunched posture, and abnormal respiration. Within the first 24-36 h, mice will begin to lose weight and likely develop ruffled coats and a hunched posture. However, if the virus is sufficiently pure, mice begin to recover, as observed by weight gain and improvement in posture and coat condition after 36 h. Mice that do not show these signs of recovery by 36 h should continue to be closely monitored for endpoint criteria. Typically, this has happened because of inaccurate tittering, potentially due to aggregation of virus particles.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63817/63817fig01.jpg" /> 
Figure 1: Infection and harvest of NDV from specified pathogen-free embryonated chicken eggs. (A) Web-like vasculature (arrows) that should be apparent after 9 days of incubation in addition to embryo movement. &#39;X&#39; denotes the interface between the chorioallantoic membrane and the air cavity, and where the hole should be created for inoculation of the egg. (B) Image depicting a dead embryo. Note the absence of weblike vasculature. A lack of embryo movement will also be observed. (C) Diagram of the components of an embryonated chicken egg showing the locations of the air cavity, yolk sac, amniotic sac, chorioallantoic membrane, and allantoic fluid. (D) Removal of the shell to expose the choriallantoic membrane. (E) Illustrates how the allantoic fluid and embryo should look following the opening of the chorioallantoic membrane. Abbreviation: NDV = Newcastle disease virus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63817/63817fig02.jpg" /> 
Figure 2: Schematic outlining the general assembly of the apparatus used for depth filtration. Ensure the pressure gauge is installed upstream of the depth filter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63817/63817fig03.jpg" /> 
Figure 3: Tangential flow filtration setup in its different configurations. Arrows show the direction of fluid flow. (A) Illustration of how the TFF cassette is assembled. (B) Schematic of the TFF system in its &#34;open&#34; configuration used during purification and concentration of fluid. (C) Schematic of the TFF setup when fluid is being eluted from the system, as used during elution of virus and cleaning solution. (D) Schematic of the TFF system in its &#34;closed&#34; configuration, which is used during cleaning of the TFF system. Abbreviation: TFF = Tangential flow filtration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63817/63817fig04.jpg" /> 
Figure 4: Density gradient ultracentrifugation and dialysis of concentrated virus from TFF. (A) Schematic showing the composition of the iodixanol density gradient. (B) Virus banding pattern following ultracentrifugation of virus produced using ML buffer during the purification process. The main virus-containing band is denoted by a black oval. (C) Typical size of a dialysis cassette loaded with 10 mL of virus prepared through an iodixanol gradient at the end of the dialysis procedure. Abbreviation: TFF = Tangential flow filtration; ML = Mannitol-Lysine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63817/63817fig05.jpg" /> 
Figure 5: Coomassie-stained SDS-PAGE gel. Gel compares the presence of contaminating proteins between unpurified (lane 2) and purified (lane 3) mesogenic NDV stocks when 1 &#215; 105 PFU were loaded. Lanes 1 and 4 = molecular weight ladder (MW). NDV HN, F0, and its subunits following cleavage, F1 and F2, along with their respective molecular weights27 are shown in white font. Abbreviations: SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NDV = Newcastle disease virus; PFU = plaque-forming units; HN = hemagglutinin; F0 = Fusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63817/63817fig06.jpg" /> 
Figure 6: Example of a 96-well plate layout used to determine virus titer by TCID50. Plate is divided into 12 columns, with each column representing a different serial dilution. Positive wells (as determined by either CPE, transgene expression, or IFA) are indicated in gray. Given the number of positive wells in this example, the expected titer after using the Spearman-Karber titer calculator (Table 2) is listed in both TCID50/mL and PFU/mL. Abbreviations: TCID50 = median tissue culture infectious dose; CPE = cytopathic effect; IFA = immunofluorescence assay; PFU = plaque-forming units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63817/63817fig07.jpg" /> 
Figure 7: Comparison of the CPE following infection of DF1 cells with lentogenic or mesogenic NDV. An MOI of 0.5 was used after 10 h of incubation (A-D) or 24 h of incubation (E-H) under different culture conditions of DMEM + 15% FBS, DMEM + 15% FBS + 5% allantoic fluid, or DMEM + 2% FBS + 125 &#181;g/mL trypsin. Scale bars = 200 &#181;m. Abbreviations: CPE = cytopathic effect; NDV = Newcastle disease virus; MOI = multiplicity of infection; FBS = fetal bovine serum; DMEM = Dulbecco&#39;s Modified Eagle&#39;s medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63817/63817fig08.jpg" /> 
Figure 8: An example of a situation where IFA is needed to titer a lentogenic NDV lacking a GFP transgene. Vero cells were cultured in DMEM containing 2% FBS and 125 &#181;g/mL trypsin. Images were taken from the 10-7 serial dilution. (A) Brightfield image of infected cells. (B) Illustrates the typical appearance of stained cells when IFA is used for detecting virus. (C) A merged image of (A) and (B). Scale bars = 100 &#181;m. Abbreviations: IFA = immunofluorescence assay; NDV = Newcastle disease virus; GFP = green fluorescent protein; FBS = fetal bovine serum; DMEM = Dulbecco&#39;s Modified Eagle&#39;s medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: PCR and RT-qPCR primers for detection of Newcastle disease virus gene segments. Primer sets used for the specific amplification of two regions of the NDV genome. F protein primer set results in the amplification of a region of the F protein that spans the F protein cleavage site. Transgene primer set amplifies the intergenic region between the phosphoprotein and matrix genes, the optimal transgene insertion site. This table also contains the primer sequences that target the viral L gene21 and the L gene probe used in the qRT-PCR assay. Abbreviations: PCR = polymerase chain reaction; RT-qPCR = reverse-transcription quantitative PCR; NDV = Newcastle disease virus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/Table 1. PCR and RT-qPCR primers_April 27 2022.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Spearmann-Karber titer calculator. This interactive spreadsheet contains the appropriate workflow and equations for determining virus concentration using TCID50 results based on well inoculum in mL, dilution scheme, and the number of replicates per dilution. Concentration is reported as volume in mL required to infect 50% of tissue culture (TCID50) and plaque-forming units per mL of virus suspension. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/Table 2. Spearmann-Karber titer calculator.xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Figure S1: Determination of iodixanol concentrations. (A) Standard curve generated from the absorbance of solutions of known iodixanol concentration at 340 nm. (B) The standard curve and equation from (A) were used to determine the concentration of iodixanol in solution following density gradient ultracentrifugation, dialysis, and polyethylene glycol concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/Supplemental Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Amplification and standard curve generated from qRT-PCR assay of synthetic 500 bp double-stranded DNA fragment of the NDV L gene. The amplification (A) and standard curve (B) were created from samples containing tenfold serial dilution (1.0 &#215; 109 copies to 1.0 &#215; 100 copies) of the DNA fragment. For the amplification and standard curve, the samples containing 1.0 &#215; 101 copies and 1.0 &#215; 100 copies were below threshold and did not produce a crossing point value below 35 Cp. The final standard curve was generated based on samples containing 1.0 &#215; 109 copies to 1.0 &#215; 102 copies of the DNA fragment. (C) DNA sequence of the 500 nucleotide NDV L gene fragment used to generate the standard curve in the qRT-PCR protocol. Abbreviations: PCR = polymerase chain reaction; RT-qPCR = reverse-transcription quantitative PCR; NDV = Newcastle disease virus; Cp = crossing point. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/Supplemental Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplemental Figure S3: Loading and extraction of NDV from the dialysis cassette. (A) The typical size of a dialysis cassette after being loaded with approximately 10 mL of virus-containing solution isolated from a sucrose density gradient and imaged at the end of the dialysis step. (B) An example of the results obtained when using iodixanol density gradient ultracentrifugation without ML buffer supplementation. Encircled is the target virus band for removal. Indicated by the arrows is an additional band that may contain damaged virions. Note that following incorporation of ML buffer in the purification process, this band is no longer apparent. Abbreviations: NDV = Newcastle disease virus; ML = mannitol-lysine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/Supplemental Figure 3.zip" target="_blank">Please click here to download this File.</a>
Supplemental Table S1: Provides the steps required for generating western blot solutions and ML buffer used during the purification process. Abbreviations: SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEMED = tetramethylethylenediamine; ML = Mannitol-lysine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63817/63817_Supp Table S1.zip" target="_blank">Please click here to download this Table.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

3.5K Views.  University of Guelph. Dit protocol schetst de technieken die worden gebruikt om Newcastle Disease Virus te produceren, zodat het veilig kan worden toegediend aan muizen en andere dieren zoals hamsters en schapen. Het gebruik van tangentiële stroomfiltratie maakt het mogelijk om grote startvolumes efficiënter te verwerken in vergelijking met bestaande procedures die alleen centrifugatieprocessen gebruiken. Dit is een langdurige procedure met verschillende ingewikkelde stappen, wat betekent dat tijdbeheer een prob...

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Immunology and Infection

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Medicine

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Bioengineering

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Streamlined Preparation of 4&#8212HAU Antigen for Accurate NDV Antibody Detection

Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article ...

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

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