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 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><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 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 ...

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Research

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Cancer Research

3.5K Views.  University of Guelph. Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields > 6 × 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.

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

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 ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

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 ...