The authors want to thank past and present members in the Adolfo Garc&iacute;a-Sastre and Peter Palese laboratories for the development of influenza reverse genetics techniques and plasmids. Research in AG-S laboratories is partially funded by CRIP, an NIAID-funded Center of Excellence for Influenza Research and Surveillance (HHSN266200700010C) and by NIAD grants R01AI046954, U01AI070469 and P01AI058113. Research in LM-S laboratory is partially funded by NIAID grant RO1AI077719.

1. Influenza virus rescue transfection
Influenza A virus belongs to the Orthomyxoviridae family of negative-stranded RNA enveloped viruses. The influenza A virus genome consists of eight different RNA genes of negative polarity that encode, at least, 11 viral proteins (Figure 1) 4. We will focus, in this report, on the rescue of one of the most common laboratory strain, influenza A/PR/8/34, 5 using ambisense plasmids (pDZ) containing the 8 influenza A/PR/8/34 vira...

<ol>
	<li>Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+influenza+A+viruses+entirely+from+cloned+cDNAs.">Generation of influenza A viruses entirely from cloned cDNAs.</a> Proc Natl Acad Sci U S A. 96, 9345-9350 (1999).</li><li>Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G., Garcia-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+influenza+A+virus+from+recombinant+DNA.">Rescue of influenza A virus from recombinant DNA.</a> J Virol. 73, 9679-9682 (1999).</li><li>Martinez-Sobrido, L., Garcia-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+influenza+virus+vectors.">Recombinant influenza virus vectors.</a> Future Virology. 2, 401-416 (2007).</li><li>Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthomyxoviridae.+The+viruses+and+their+replication.">Orthomyxoviridae. The viruses and their replication.</a> Fields Virology. , 1647-1689 (2006).</li><li>Schickli, J. H., Flandorfer, A., Nakaya, T., Martinez-Sobrido, L., Garcia-Sastre, A., 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=Plasmid-only+rescue+of+influenza+A+virus+vaccine+candidates.">Plasmid-only rescue of influenza A virus vaccine candidates.</a> Philos Trans R Soc Lond B Biol Sci. 356, 1965-1973 (2001).</li><li>Quinlivan, M., Zamarin, D., Garcia-Sastre, A., Cullinane, A., Chambers, T., 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=Attenuation+of+equine+influenza+viruses+through+truncations+of+the+NS1+protein.">Attenuation of equine influenza viruses through truncations of the NS1 protein.</a> J Virol. 79, 8431-8439 (2005).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li></ol>

Rescue of recombinant influenza viruses from plasmid DNA is a simple and straightforward process once the protocol is routinely performed in the laboratory, but in the beginning, multiple things can go wrong. It is imperative to have good plasmid preparation to generate the virus. Proper maintenance of the cell lines (293T and MDCK) is crucial for a successful viral rescue. Traditionally, a genetic tag is inserted into an influenza gene-encoding plasmid, by silent mutagenesis. Introduction of this silent mutation(s) and ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-065</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>OptiMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>51985-034</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Lipofectamine 2000 (LPF2000)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>TPCK-trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T-8802</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>Bovine Albumin (BA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7979</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300-054</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>Penicillin/Streptomycin (PS) 100X</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS) </td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30070.03</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>V-bottom 96-weel plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>249570</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Cell lines
293T (catalogue number CRL-11268) and MDCK (catalogue number CCL-34) cell lines are maintained in a 37&deg;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. Cells are available form the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA).
Embryonated chicken eggs
Embryonated 10-day-old 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. Eggs are incubated at 37&deg;C preceding and after viral infection. Before and after viral infection, eggs are candled to determine viability of the embryos. It is very important to look for dead eggs before and after viral infection. Before infection a dead egg can be easily spotted by the absence of blood vessels as well as the absence of embryo mobility. When candled, live embryos move. After viral infection a dead egg (probably related to influenza virus infection) will be easily spotted by the bad appearance of the egg as seen by the smaller and bloody volume of allantoic fluid. Infected-eggs are discarded in double autoclavable bags and autoclaved following standard procedures.
Chicken red blood cells (RBC)
Chicken RBC can be purchased from Truslow Farms, 201 Valley Road, Chestertown, Md 21620. Store at 4&deg;C. For HA assays, wash 5 ml of the chicken RBC with 45 ml of PBS 1X in a 50 ml centrifuge tube. Centrifuge for 5 minutes at 1000 rpms, RT. Discard carefully the supernatant and use a 1:1000 dilution of the pelleted RBC in PBS 1X (final concentration of 0.5-1.0% RBC).
Tissue culture supernatants and allantoic fluids
Both, tissue culture supernatants and allantoic fluids can be stored at 4&deg;C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80&deg;C.
Plasmids
All plasmids are prepared using a plasmid maxi kit following manufacturer's recommendations. All plasmids are aliquot at concentrations of 1 &mu;g/ml in ddH2O and stored at -20&deg;C. For short-term storage, the plasmid can be keep at 4&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 appropriated for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel chromatography. Ambisense pDZ plasmids (6) containing the eight influenza A/PR/8/34 viral genes (7) are illustrated in Figure 2.
Viruses
The described protocol for rescuing influenza A/PR/8/34 can be performed under biosafety level (BSL) 2 conditions. Contaminated material, including tissue culture supernatants and embryonated eggs, should be sterilized before disposal. Rescue of other influenza virus may require higher BSL conditions and, therefore, special conditions/security measurements will need to be followed.
Tissue culture media and solutions
DMEM 10%FBS 1%PS: 445 ml Dulbecco's modified Eagle's medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4&deg;C. This media will be used to maintain 293T and MDCK cells as well as for the transfections. DMEM 0.3%BA 1%PS: 495.7 ml of DMEM, 4.3 ml of 35% Bovine Albumin (BA). Store at 4&deg;C. Just before use, add TPCK treated trypsin to a final concentration of 1 &mu;g/ml. Infectious media.
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 liter. 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.		</div>

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.

Watch this Scientific Journal Video about Generation of Recombinant Influenza Virus from Plasmid DNA at JoVE.com

Generation of Recombinant Influenza Virus from Plasmid DNA

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 ...

generation-of-recombinant-influenza-virus-from-plasmid-dna

Research

JoVE Journal

Immunology and Infection

30.0K Views.  University of Rochester School of Medicine and Dentistry. Salvataggio di virus dell'influenza A dal DNA plasmide è una tecnica di base ed essenziale sperimentale che consente ai ricercatori di generare virus influenzali ricombinanti per studiare i molteplici aspetti della biologia del virus dell'influenza, e da utilizzare come potenziali vettori o vaccini.