We thank past and present members in JCT and LM-S laboratories for the development and improvement of the arenavirus reverse genetics techniques and plasmids. We also thank Snezhana Dimitrova for technical support. The monoclonal antibody directed to JUNV NP (SA02-BG12) was obtained from BEI Resources (NIAID Biodefense and Emerging Infections Research Resources Repository). BYHC was supported by Grant Number GM068411 from Institutional Ruth L. Kirschstein National Research Service Award. EO-R is a Fullbright-Conicyt (BIO 2008) and a Rochester Vaccine Fellowship (2012) recipient. EO-R current support is provided by a postdoctoral Fellowship for Diversity &amp; Academic Excellence from the Office for Faculty Development &amp; Diversity at the University of Rochester. Research in LM-S laboratory is funded by the NIH grants RO1 AI077719, R21NS075611-01, R03AI099681-01A1, the NIAID Centers of Excellence for Influenza Research and Surveillance (HHSN266200700008C), and The University of Rochester Center for Biodefense Immune Modeling (HHSN272201000055C).
Research at JCT laboratory was supported by grants RO1 AI047140, RO1 AI077719, and RO1 AI079665 from NIH.

1. Arenavirus Rescue Transfection
Our rescue system was based on the use of both polymerase II protein expression plasmids encoding the nucleoprotein (NP) and RNA-dependent-RNA-polymerase (L), the viral trans-acting factors required for RNA replication and gene expression of the arenavirus genome 7, 19 , and plasmids capable to direct intracellular synthesis, via the cellular RNA polymerase I (pol-I), of the S and L antigenome RNA species 7 . In our studies we use the pCAGGs protein expression plasmid, which uses the chicken &beta;-actin promoter and polyadenylanation (pA) signal sequences, and the human pol-I plasmid, which uses the human polymerase I promoter and murine terminator sequences (Figure 2). The S and L RNA segments are cloned into the hpol-I plasmid in an antigenomic orientation to allow for the generation of genomic RNA segments upon transcription by hpol-I. For the rescue of wild-type recombinant LCMV (rLCMV) and Candid#1 (rCandid#1) the pCAGGs NP and L from each virus were co-transfected together with their respective human pol-I S and L RNA segments (Figure 3). Generation of recombinant trisegmented LCMV (r3LCMV) and Candid#1 (r3Candid#1) followed a similar protocol but the S segment was split into two different pol-I S plasmids, each encoding a distinctive reporter gene: in one, the NP open reading frame (ORF) was replaced with the Green Fluorescent Protein (GFP) reporter gene (hpol-I S GFP/GP) and, in the other, the viral glycoprotein precursor (GPC) ORF is replaced with the Gaussia luciferase (Gluc) reporter gene (hpol-I S NP/Gluc). A schematic representation of the protocol is illustrated in Figure 4. The following transfection and infection protocol has been established for 6-well-plates.
<ol>
	<li>OptiMEM-Lipofectamine 2000 (LPF2000) mixture: Prepare 250 &mu;l of OptiMEM media and, depending on the virus rescue, 10 - 12 &mu;g of LPF2000 (1 &mu;g/&mu;l) per transfection (Table 1). A ratio of 2.5 &mu;g LPF2000 per &mu;g of DNA is recommended. Incubate for 5 - 10 min at RT. Meanwhile, prepare the plasmid transfection mixture in a separate microcentrifuge tube (step 1.2).</li>
	<li>Plasmid DNA transfection mixture: Prepare the plasmid transfection cocktail in a microcentrifuge tube using the recommended amounts for each virus rescue (see Table 1). Bring the final volume to 50 &mu;l with OptiMEM.</li>
	<li>OptiMEM-LPF2000-DNA plasmid mixture: Add 250 &mu;l of the OptiMEM-LPF2000 mixture (step 1.1) into the plasmid DNA transfection mixture (step 1.2). Incubate this mixture for 20 - 30 min at RT. Meanwhile prepare and count 1 x 106 Vero cells per transfection reaction. Vero cells will be transfected in suspension.</li>
	<li>Preparation of Vero cells: Vero cells are cultured in 100 mm dishes. Before starting, bring the 1x PBS, DMEM 10% FBS 1% PS media, and trypsin-EDTA mixture to 37 &deg;C.
	<ol>
		<li>Wash cells, twice, with 5 ml of 1x PBS.</li>
		<li>Trypsinize cells with 1 ml of trypsin-EDTA and wait until cells detach (approximately 5 min). Gentle tapping maybe required to fully detach the cells from the dishes.</li>
		<li>Carefully, resuspend the cells in 10 ml of DMEM 10% FBS 1% PS in a 15 ml centrifuge tube.</li>
		<li>Centrifuge the cells for 5 min at 1,000 rpm in a centrifuge.</li>
		<li>Resuspend the cells in 10 ml of DMEM 10% FBS 1% PS and count the cells using a hemocytometer and adjust concentration to 1 x 106 cells/ml. It was observed that 0.5 - 1 x 106 Vero cells per transfection produced the best results.</li>
	</ol>
	</li>
	<li>Mix LPF2000/DNA and cells: After 20 - 30 min room temperature incubation (step 1.3), add 1 ml of Vero cells (1 x 106) (step 1.4) to the OptiMEM-LPF2000-DNA plasmid mixture and incubate for 5 min at room temperature.</li>
	<li>Seed LPF2000/DNA-cell mix into 6-well-plates: Add the DNA-LPF2000-Vero mixture (step 1.5) into the wells of the 6-well plate. Gently shake the 6-well-plate and let the transfection incubate overnight in the incubator at 37 &deg;C and 5% CO2.</li>
	<li>Change transfection medium: Approximately 16 - 24 hr post-transfection, remove the transfection media, add 2 ml of infection media and incubate transfected cells for an additional 48 hr.</li>
	<li>Cell passage: After 48 hr of incubation in infection media, remove the tissue culture supernatant (TCS) and pass the transfected cells into a 100 mm dish. The TCS rarely contains infectious virus because the transfected cells need additional incubation to generate virus particles. For the cell passage:
	<ol>
		<li>Wash cells, twice, with 2 ml of 1x PBS.</li>
		<li>Trypsinize cells with 0.5 ml of trypsin-EDTA and wait until cells detach.</li>
		<li>Resuspend the cells in 1 ml of DMEM 10% FBS 1% PS in a 1.5 ml microcentrifuge tube.</li>
		<li>Centrifuge the cells for 5 min at 5,000 rpm, 4 &deg;C in a microcentrifuge.</li>
		<li>Carefully resuspend the cells in 1 ml of infection media and transfer the cells to a 100 mm dish. Bring up the total volume in the plate, with infectious media, to 8 ml, a sufficient volume to prevent drying of the cells as well as for concentrating the virus.</li>
	</ol>
	</li>
	<li>Gently shake the 100 mm dish and continue the incubation of Vero cells in the incubator at 37 &deg;C and 5% CO2 for 72 hr.</li>
	<li>Collection of TCS: After 72 hr incubation, collect the TCS in a 15 ml centrifuge tube. Remove cell debris by centrifuging for 5 min at 2,500 rpm in a centrifuge. Infectious arenavirus is found in the TCS, so remove from the cell debris pellet and store the TCS at -80 &deg;C.</li>
</ol>
2. Confirmation of Recombinant Arenavirus Rescue
Detection of a successful rescue of rLCMV and rCandid#1 is achieved via a focus forming unit (FFU) immunofluorescence assay (IFA). For wild-type virus rescue we use antibodies specific for the viral NP. The r3LCMV and r3Candid#1 encode two reporter genes (GFP and Gluc), thus allowing viral detection and titration without the need of antibodies by fluorescence microscopy (GFP) and/or luminescence (Gluc).
<ol>
	<li>Confirm wild-type recombinant arenavirus rescue: The day before titration, wash Vero cells twice with PBS, trypsinize and prepare 96-well plates to reach 80 - 90% confluence next day (4 x 104 cells/well). Gently shake the plates by hand to get a uniformed distribution of the cells. Culture the cells, overnight, in the 37 &deg;C incubator with 5% CO2. Check the cells under the microscope to confirm a monolayer, before proceeding with the infection:
	<ol>
		<li>Serially dilute (10 fold dilutions) the virus-containing TCS recovered from the transfected cells in the 100 mm dish (step 1.10) in OptiMEM.</li>
		<li>Remove media from the seeded Vero cells, wash twice with 50 &mu;l of 1x PBS, and infect cells with 50 &mu;l of the serially diluted virus (step 2.1). Infect cells for 1.5 hr at 37 &deg;C and 5% CO2.</li>
		<li>After infection, remove the virus inoculum, add 100 &mu;l of infection media/well and incubate the cells for 16 - 18 hr. Viral re-infection may occur after 18 hr incubation, which could result in over-estimation of the viral titers.</li>
		<li>At 16 - 18 hr post-infection, remove the TCS from each well and fix the cells with 4% formaldehyde diluted in 1x PBS, for 15 min at RT.</li>
		<li>Next, remove the fixation solution and permeabilize the cells with 0.1% triton X-100 diluted in 1x PBS, for 10 min at room temperature.</li>
		<li>Aspirate the permeabilization solution, then wash cells 3 times with 1x PBS.</li>
		<li>Block the cells with 2.5% albumin bovine serum (BSA) in 1x PBS (blocking solution) for 1 hr at RT. Alternatively, the cells can be blocked overnight at 4 &deg;C and continued with antibody incubation the following day.</li>
		<li>Meanwhile prepare the primary antibody. Dilute the LCMV- and Candid#1-specific primary antibodies in blocking solution, and then centrifuge the antibody/blocking solution for 15 min at 3,500 rpm. The monoclonal anti-LCMV NP antibody clone 1.1.3 (1:30 dilution) 16 and the monoclonal anti-JUNV NP antibody SA02-BG12 from BEI Resources (1:500 dilution) can be used for the detection of rLCMV and rCandid#1, respectively.</li>
		<li>After a 1 hr incubation, aspirate the blocking solution and add 50 &mu;l of the primary antibody to the cells and incubate for 1 hr at 37 &deg;C.</li>
		<li>At this time, dilute the secondary antibody in blocking solution, and then centrifuge the antibody/blocking solution for 15 min at 3,500 rpm. The polyclonal rabbit anti-mouse IgG FITC secondary antibody for the detection of primary monoclonal antibodies was used to obtain images observed in Figure 5.</li>
		<li>After 1 hr incubation, aspirate the primary antibody, wash 3 times with 1x PBS, and add the secondary antibody for 30 min at 37 &deg;C.</li>
		<li>Following 30 min incubation, aspirate the secondary antibody and wash 3 times with 1x PBS. The cells are now ready to be observed using fluorescence microscopy to both determine the success of viral rescue and titration by counting the focus forming units per ml (FFU/ml).</li>
	</ol>
	</li>
	<li>r3LCMV and r3Candid#1 viral rescue: Since these viruses express two reporter genes, their rescues can be monitored by fluorescence microscopy to detect GFP expression or Gluc expression in TCS. Rescue can be also confirmed by expression of Gluc in TCS. To titrate the trisegmented viruses, we follow steps 2.1 to 2.1.4, however cells do not need to be fixed to determine a successful viral rescue because of GFP expression. The titration of the virus will be determined by counting the FFU/ml. To determine successful viral rescue by Gluc expression, Gluc expression from TCS can be measured using a Gluc assay kit (Biolux Gaussia Luciferase Assay kit, New England Biolabs). To that end:
	<ol>
		<li>Pipet 100 &mu;l of TCS into a 96-well white plate (Flat bottom microtiter plates, Fisher Scientific).</li>
		<li>Set up the luminometer (LumiCount, Packard Biosciences).</li>
		<li>Add 50 - 100 &mu;l Gluc Assay solution (as recommended by the manufacturer) to each sample and measure the Gluc reporter gene expression with the luminometer. As a negative control, Gluc expression of the TCS from the wild-type viruses infected cells was measured.</li>
	</ol>
	</li>
</ol>
3. Passage of Tissue Culture Supernatants
Viral rescue depends on transfection efficiencies. Vero cells have been shown to have lower transfection efficiencies than other cell lines 14 . If virus titers in the TCS are low, infect fresh Vero cells at a multiplicity of infection (MOI) of 0.01 (LCMV) or 0.1 (Candid#1) for 72 hr to amplify the virus.

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No conflicts of interest declared.

Generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has become a widely used approach for the investigation of many different facets of arenavirus biology. Here we document a crucial improvement to the current system by performing arenavirus rescues in Vero cells, allowing for the potential generation of FDA-approved vaccine candidates against arenaviruses and vaccine vectors against other infectious diseases.
The experimental procedures involved are in gener...

Arenaviruses are enveloped viruses with a bisegmented, negative-stranded RNA genome 3 that belong to the Arenaviridae family. The arenavirus genome encodes four proteins in an ambisense fashion from two separate viral segments 3. The large (L) segment encodes the RNA-dependent RNA polymerase (L) and the small RING (Really Interesting New Gene) finger protein (Z) that serves as the main driving force of virus budding. The small (S) segment encodes the viral nucleoprotein (NP) and the surface glycoprotein (GP) (Figure 1).
Several members of the Arenaviridae family are responsible for lethal hemorrhagic fever (HF) in humans 3. Of principle concerns are Lassa virus (LASV) and Jun&iacute;n virus (JUNV), which are known to cause high mortality in hospitalized patients 8, 9 . Although these viruses are endemic to West Africa and rural Argentina, respectively, there are increasing concerns of importation of LASV and JUNV to non-endemic areas due to increased travel 10. Additionally, although not normally associated with severe disease in humans, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is considered a neglected pathogen as there have been cases of lethal infection in immunocompromised patients 6, 15 and is responsible for congenital birth defects and spontaneous abortions in pregnant women 2, 13 . Currently there are no US FDA-approved vaccines against arenaviruses and treatment is limited to the nucleoside analog ribavirin, which is only partially effective and often associated with significant side effects.
The introduction of plasmid-based reverse genetics 4 and generation of recombinant arenaviruses 7, 19 have greatly advanced the field of arenavirus research. Currently, rodent cells (such as BHK-21) are used for the generation of recombinant arenaviruses due to the species-specific murine RNA polymerase I (pol-I) promoter directing the initial transcription of the S and L segments. However virus rescue in BHK-21 cells are not an approved method for the generation of recombinant arenaviruses as potential vaccine seed candidates. Here we document the use of the human pol-I promoter for efficient rescue of the prototypic Old World (OW) LCMV and the New World (NW) JUNV Candid#1 strain in Vero cells. Using a similar methodology, we generated recombinant trisegmented LCMV (r3LCMV) and Candid#1 (r3Candid#1) arenaviruses that contain two additional foreign genes encoded in two different S RNA segments 5 . This new system not only follows a highly reproducible and simple protocol but can be immediately implemented in the generation of recombinant arenaviruses as potential vaccine or vaccine vector seeds.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Material and methods&lt;/strong&gt;</td></tr><tr><td colspan="4"> &lt;strong&gt;Cell lines&lt;br/&gt; &lt;/strong&gt;Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &amp;deg;C incubator with 5 % CO&lt;sub&gt;2&lt;/sub&gt; in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586).&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Plasmids&lt;br/&gt; &lt;/strong&gt;All plasmids, with the exception of hpol-I L, can be grown at 37 &amp;deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &amp;deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer&#039;s recommendations and stored at -20 &amp;deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Viruses&lt;br/&gt; &lt;/strong&gt;The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Tissue culture media and solutions&lt;br/&gt; &lt;/strong&gt;&lt;strong&gt;DMEM 10 %FBS 1 %PS:&lt;/strong&gt; 445 ml Dulbecco&#039;s modified Eagle&#039;s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &amp;deg;C. This media will be used for maintenance of Vero cells.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Infectious Media:&lt;/strong&gt; 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &amp;deg;C. This media will be used during viral infections.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;10X Phosphate buffered saline (PBS):&lt;/strong&gt; 80 g of NaCl, 2 g of KCl, 11.5 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O, 2 g of KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;. Add ddH&lt;sub&gt;2&lt;/sub&gt;O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature.&lt;br/&gt; &lt;strong&gt;1X PBS:&lt;/strong&gt; Dilute 10X PBS 1:10 with ddH&lt;sub&gt;2&lt;/sub&gt;O. Sterilize by autoclave and store at room temperature.&lt;br/&gt; &lt;strong&gt;2.5 % BSA&lt;/strong&gt;: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &amp;deg;C. This is used as a blocking solution for IFA. </td></tr></tbody></table>

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.

Successful rescue of a recombinant wild-type arenavirus will be confirmed by the presence of viral antigens using IFA (Figure 5). In the case of recombinant trisegmented viruses, successful viral rescue can be assessed by observing GFP expression by fluorescence microscopy (Figure 6A). Successful rescue will be further confirmed by assessing Gluc expression (Figure 6B). Representative results illustrating the successful rescue of wild-type and trisegmented arenavirus wer...

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Generation of Recombinant Arenavirus for Vaccine Development 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 ...

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Immunology and Infection

16.9K Views.  University of Rochester School of Medicine and Dentistry. 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 v...

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

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 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.

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 ...

Cancer Research

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

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

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