Name: rescue of recombinant newcastle disease virus from cdna
Uploaded: 2013-10-11T23:30:00
Description: Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Authors would like to thank past and present members in the laboratories of Drs. Peter Palese and Adolfo Garc&iacute;a-Sastre for the development of NDV reverse genetics techniques and for technical assistance. Research in Newcastle disease virus in AG-S laboratory is partially funded by NIAD grant R01AI088770 and by the Department of Homeland Security Science &amp; Technology Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD, award number 2010-ST-061-AG001). 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).

1. Preparation of Mammalian Cells (Figure 4A, Day 1)
Split HEp-2 or A549 cells the day before transfection in 6-well plates. Density of the cells should reach 80-90% confluence the following day. Usually, a confluent 100 mm dish can be split into 8 wells (around 1 x 106 cells per well). For each virus to be rescued, 2-4 different wells should be included, as well as 2 extra wells for the controls pCAGGs-GFP and pCITE-GFP18, aimed to monitor transfection and...

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Virol. 80 (Pt 11), 2987-2995 (1999).</li><li>Nakaya, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Newcastle+disease+virus+as+a+vaccine+vector.">Recombinant Newcastle disease virus as a vaccine vector.</a> J. Virol. 75, 11868-11873 (2001).</li><li>Zamarin, D., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+Newcastle+disease+virus+for+cancer+therapy:+old+challenges+and+new+directions.">Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions.</a> Future Microbiol. 7, 347-367 (2012).</li><li>Park, M. S., Garcia-Sastre, A., Cros, J. F., Basler, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+V+protein+is+a+determinant+of+host+range+restriction.">Newcastle disease virus V protein is a determinant of host range restriction.</a> J. Virol. 77, 9522-9532 (2003).</li><li>Park, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+(NDV)-based+assay+demonstrates+interferon-antagonist+activity+for+the+NDV+V+protein+and+the+Nipah+virus+V,+W,+and+C+proteins.">Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins.</a> J. Virol. 77, 1501-1511 (2003).</li><li>Zamarin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+oncolytic+properties+of+recombinant+newcastle+disease+virus+through+antagonism+of+cellular+innate+immune+responses.">Enhancement of oncolytic properties of recombinant newcastle disease virus through antagonism of cellular innate immune responses.</a> Mol. Ther. 17, 697-706 (2009).</li><li>Vigil, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+reverse+genetics+to+enhance+the+oncolytic+properties+of+Newcastle+disease+virus.">Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus.</a> Cancer Res. 67, 8285-8292 (2007).</li><li>Swayne, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+paramyxovirus+type+1-avian+influenza-H7+virus+as+a+vaccine+for+protection+of+chickens+against+influenza+and+Newcastle+disease.">Recombinant paramyxovirus type 1-avian influenza-H7 virus as a vaccine for protection of chickens against influenza and Newcastle disease.</a> Avian Dis. 47, 1047-1050 (2003).</li><li>Park, M. S., Steel, J., Garcia-Sastre, A., Swayne, D., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+viral+vaccine+constructs+with+dual+specificity:+avian+influenza+and+Newcastle+disease.">Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease.</a> Proc. Natl. Acad. Sci. 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Virol. 83, 584-597 (2009).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+type+I+interferon+secretion+through+recombinant+Newcastle+disease+virus+expressing+measles+virus+hemagglutinin+stimulates+antibody+secretion+in+the+presence+of+maternal+antibodies.">Induction of type I interferon secretion through recombinant Newcastle disease virus expressing measles virus hemagglutinin stimulates antibody secretion in the presence of maternal antibodies.</a> J. Virol. 85, 200-207 (2011).</li><li>DiNapoli, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus,+a+host+range-restricted+virus,+as+a+vaccine+vector+for+intranasal+immunization+against+emerging+pathogens.">Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 9788-9793 (2007).</li><li>Martinez-Sobrido, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+respiratory+syncytial+virus+by+a+recombinant+Newcastle+disease+virus+vector.">Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector.</a> J. Virol. 80, 1130-1139 (2006).</li><li>Bukreyev, A., Collins, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus+as+a+vaccine+vector+for+humans.">Newcastle disease virus as a vaccine vector for humans.</a> Curr. Opin. Mol. Ther. 10, 46-55 (2008).</li><li>Martinez-Sobrido, L., Zuniga, E. I., Rosario, D., Garcia-Sastre, A., de la Torre, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+type+I+interferon+response+by+the+nucleoprotein+of+the+prototypic+arenavirus+lymphocytic+choriomeningitis+virus.">Inhibition of the type I interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus.</a> J. Virol. 80, 9192-9199 (2006).</li><li>Jennings, S., Martinez-Sobrido, L., Garcia-Sastre, A., Weber, F., Kochs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thogoto+virus+ML+protein+suppresses+IRF3+function.">Thogoto virus ML protein suppresses IRF3 function.</a> Virology. 331, 63-72 (2005).</li><li>Kochs, G., Garcia-Sastre, A., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+anti-interferon+actions+of+the+influenza+A+virus+NS1+protein.">Multiple anti-interferon actions of the influenza A virus NS1 protein.</a> J. Virol. 81, 7011-7021 (2007).</li><li>Munoz-Jordan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+alpha/beta+interferon+signaling+by+the+NS4B+protein+of+flaviviruses.">Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses.</a> J. Virol. 79, 8004-8013 (2005).</li><li>Cardenas, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ebola+virus+VP35+protein+binds+double-stranded+RNA+and+inhibits+alpha/beta+interferon+production+induced+by+RIG-I+signaling.">Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling.</a> J. 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Several factors are to be considered to achieve good results while rescuing NDV. First, the full-length cDNA construct to be used needs to be designed to allow the functional incorporation of the new transgenes/modifications into the NDV genome. This means, as stated above, that (i) appropriate gene end (GE), intergenic (IG) and gene start (GS) sequences are to be added if required; (ii) there are no putative GE or GS sequences into the foreign gene, and (iii) the full recombinant genome follows the &#34;rule of six&#34;...

Newcastle disease virus (NDV), an avian paramyxovirus belonging to the Avulavirus genus1, is an economically relevant and thus widely researched and surveilled zoonotic agent, which can severely affect poultry farming all around the world. Although not a human pathogen, NDV has also been thoroughly studied beyond the veterinarian field both as a model paramyxovirus and due to its highly interesting, natural oncolytic properties6. Research on NDV greatly benefited from the development of reverse genetics techniques for single-stranded, non-segmented negative-sense RNA viruses, first described for rabies virus by Conzelmann and coleagues2. A variety of genetically modified NDVs, carrying foreign genes or modifications of their wild type genome have been widely studied ever since. Work with these recombinant viruses has been critical to characterize different virulence factors not only of NDV but also of other relevant human pathogens such as influenza A virus7 - or the emergent Nipah virus8. Furthermore, a number of different studies have explored the use of these techniques to improve the innate antitumoral activity of NDV6,9,10, mostly by enhancing the immunostimulatory properties of the virus. Other relevant area of research on recombinant NDVs has been the generation of vaccine candidates against other viral diseases such as influenza5,11,12, HIV13, measles14, SARS15, or that caused by the respiratory sincytial virus (RSV)16. Amongst the various noteworthy advantages provided by NDV are the lack of preexisting immunity in human populations, the stability of the foreign genetic inserts, a lack of recombinatory activity and overall a high safety profile combined with the aforementioned natural immunostimulatory properties17. It&#39;s also noteworthy the potential use of recombinant bivalent vaccines in poultry, protective against both NDV and highly pathogenic avian influenza viruses11,12. This may be an excellent way to decrease the chances of the latter spreading from wild to domesticated animals, thus also helping to prevent a possible inter-specific jump of the dreaded avian influenza to humans. Finally, reporter-expressing NDV have been used for the evaluation of innate immune responses as well as the identification of interferon antagonist encoded by multiple viruses18-27.
The rescue process of a recombinant, non-segmented, negative-stranded RNA virus basically consists on artificially forcing a viral replication cycle in a producing cell by transfecting cDNA encoding the minimal infective molecular machinery, known as ribonucleoprotein or RNP (Figure 2). The RNPs consist of the viral polymerase (P and L proteins), the nucleoprotein (NP) and the full-length antigenomic RNA of the virus. This RNA+ antigenome is the template required for the generation of the complementary RNA- genomes, which, also associated with the rest of the proteins of the viral RNP, recapitulates the same infectious complex that a natural virus would release on the cytoplasm of the cell upon infection (Figure 2A). From this step onwards, the viral cycle can proceed naturally and recombinant virions, encapsidating the modified genomes, will be generated (Figure 2B). Remarkably, transfection of the genomic cDNA instead of the antigenomic cDNA greatly impairs or completely abolishes rescue efficiency2,28-30. Even when antigenomic cDNA is transfected, the efficiency of the encapsidation of the recombinant RNA into RNPs in transfected cells is probably very low. Because of this, rescue protocols for NDV often include different steps for the amplification of the few viral particles released from the originally transfected cells by coculturing them with permissive cells and/or by the infection of embryonated eggs.
Prior to the rescue, the cDNA can be manipulated by standard cloning procedures in order to generate the desired modifications. While specific mutations of the different gene products and regulatory sequences of the virus can be straightforwardly achieved this way, many of the published work involving recombinant NDV has required the addition of a new transcriptional unit into the NDV genome. Like other members of the paramyxovirus family, the NDV genome encodes eight different proteins into six transcriptional units, which are differentially expressed depending on their location respect to the 3&#39; end in a decreasing gradient critical for the viral life cycle1. Because of this, the location of the new transcriptional unit within the genome must be carefully chosen to achieve a balance between expression of the transgene and impairment of viral replication. Insertion between P and M genes has been used the most, though other sites have also been tested13,31.
Whatever the insert, the cloning into NDV cDNA needs to follow some rules to generate a rescuable construct: (i) any new gene to be included into the NDV genome has to be under control of the appropriate signals for the viral RNA-dependent-RNA polymerase. These sequences must be added upstream of the new open reading frame (ORF) so the polymerase can recognize the end of the previous gene (GE) and the beginning of the new transgene (GS), spaced by a single nucleotide intergenic sequence (IG). Addition of a valid Kozak (K) sequence to improve eukaryotic ribosomal translation is also recommended for better foreign protein expression32; (ii) efficient replication of NDV, as for most members of the Paramyxoviridae family, is dependent on the genome length being multiple of six33; therefore, any insertion into the NDV has to follow this &#34;rule of six&#34;. If necessary, required additional nucleotides can be added downstream the new ORF; and (iii) the sequence of the transgene should be checked to find possible GE and GS like sequences which could impair rescue efficiency, transgene expression and/or virus viability. If present, these sequences must be removed by silent mutagenesis. The generation of recombinant full length cDNA following aforementioned rules is the first step in order to efficiently produce a genetically modified NDV as detailed here.
In the system all DNA constructs are under control of the T7 RNA polymerase promoter (Figure 3). This cytoplasmic polymerase is provided in trans by coinfection with a recombinant modified vaccinia Ankara virus (MVA-T7)34. Figure 3A show the pNDV-B1 plasmid, which encodes the full-length antigenomic cDNA5. Figure 3B shows pTM1 plasmids encoding NP, P and L ORFs. Plasmids pCITE-GFP, which encodes, under the T7 promoter, the Green Fluorescent Protein (GFP), and pCAGGs GFP18, which encodes the same ORF under the chicken beta actin promoter 35, are used as controls. In this protocol we show the procedure to rescue recombinant NDV from the cDNA of the lentogenic NDV strain Hitchner B15 (Figure 4).

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

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.

Rescue of NDV is a well-established procedure, routinely performed in the laboratories that have access to the complete cDNA of the virus. However, the intrinsic stochastic nature of the method makes it difficult to achieve 100% rescue efficiency. Monitoring the early steps of the process, specially the transfection efficiency and the infection with MVA-T7, helps identifying possible problems. Figure 5A shows standard transfection and transfection/infection efficiencies that are enough for a successful N...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

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, ...

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