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 viral segments (Figure 2).
For the rescue of recombinant influenza viruses from plasmid DNA , we recommend 3 independent transfections per each recombinant virus. If more than one recombinant virus rescue is attempted, scale the following steps accordantly to the number of viruses to be rescued. The following transfection and infection protocol is established for 6-well-plates. A schematic representation of the protocol is illustrated in Figure 3.
<ol>
 <li>OptiMEM-Lipofectamine 2000 (LPF2000) mixture: Prepare 250 &mu;l of OptiMEM media and 6-8 &mu;l of LPF2000 per transfection. Incubate for 5-10 minutes at room temperature (RT). Meanwhile, prepare the plasmid transfection mixture.</li>
 <li>Plasmid transfection mixture: Prepare the plasmid transfection cocktail in 50 &mu;l of OptiMEM media. We usually use 1 &mu;g of each influenza DNA plasmid per rescue. Add 1 &mu;l of the pDZ plasmids (at 1 &mu;g/&mu;l) PB2, PB1, PA, HA, NP, NA, M, and NS to a tube containing 50 &mu;l of OptiMEM media.</li>
 <li>OptiMEM-LPF2000-DNA plasmid mixture: Add 250 &mu;l from step 1.1 into the influenza DNA plasmid transfection mixture (step 1.2). Incubate this mixture for 20-30 minutes at RT. Meanwhile, prepare suspensions of 293T and MDCK cells for transfection.</li>
 <li>Preparation of 293T/MDCK co-culture: Before starting, bring the PBS 1X, DMEM 10%FBS 1% PS media, and EDTA-trypsin mixture to 37&deg;C. The density of the cells should be at 80-90% confluence the day of transfection. Usually, one confluent 100 mm dish of 293T and one confluent 100 mm dish of MDCK cells can be used for 10-12 rescues. We are going to use 250 &mu;l of cells per well. Both cell lines will be resuspended in a total of 3 ml of DMEM 10%FBS 1%PS.
 <ul>
 <li>Carefully resuspend each cell line in 10 ml of DMEM 10%FBS 1%PS in a 15 ml centrifuge tube. You will have one tube for 293T cells and one tube for MDCK cells.</li>
 <li>Resuspend the 293T cells in 3 ml of DMEM 10%FBS 1%PS and when resuspended, deliver the 3 ml to the MDCK cells to resuspend those cells. This will give you the mixture of 293T and MDCK cells to be used for your co-culture.</li>
 <li>Add 250 &mu;l of the 293T/MDCK cells per well (10-12 6-well wells).</li>
 </ul>
 </li>
 <li>After 20-30 minutes RT incubation (step 1.3), add 1 ml of DMEM 10%FBS 1%PS to the OptiMEM-LPF2000-influenza DNA plasmid mixture.</li>
 <li>Add the 1.3 ml (step 1.5) into the wells with the 250 &mu;l of 293T/MDCK cells (step 1.4).</li>
 <li>Gently shake the 6-well-plate and let the transfection incubate overnight (ON) in the incubator at 37&deg;C and 5% CO2.</li>
 <li>Next day, approximately 16-24 hours post-transfection, change the transfection media and incubate the transfected cells in DMEM 0.3%BA 1%PS containing 1 &mu;g/ml of TPCK-trypsin for 48 hours.</li>
 <li>After 48 hours of changing the media, transfer the supernatant from the transfected cells into a microcentrifuge tube.</li>
 <li>Centrifuge the tissue culture supernatant in a microcentrifuge for 1-2 minutes, 13,000 rpm.</li>
 <li>Infect fresh MDCK cells in 6-well plates (plated the day before) or 10-day-old chicken embryonated eggs with 200 &mu;l of centrifuged tissue culture supernatants from step 1.10. Incubate the cells and/or eggs at 37&deg;C for 2-3 days.
 <ol>
 <li>Infection of 10-day-old chicken embryonated eggs: All procedures to infect chicken embryonated eggs are performed under sterile conditions.
 <ol>
 <li>Candle the 10-day-old eggs using a light-candling box to see the interface between the air sac and the allantoic cavity. Make a pencil mark on the interface border.</li>
 <li>With a 5 ml syringe needle make a hole in the eggshell.</li>
 <li>With a 1 ml syringe, infect each egg with 200 &mu;l of the tissue culture supernatants from step 1.10.</li>
 <li>Cover the hole in the eggshell with melted wax using a cotton swab.</li>
 <li>Incubate the infected eggs at 37&#959;C for 2-3 days.</li>
 </ol>
 </li>
 <li>Infection of fresh MDCK cells: The day before passage the tissue culture supernatant from the 293T/MDCK co-cultures, prepare 6-well plate dishes with MDCK cells to reach 80-90% confluence next day. Usually, a confluent 100 mm tissue culture plate can be split into 6-8 wells. Wash the cells, twice, with PBS 1X, trypsinize and prepare the 6-well plates. Gently shake by hand the plates in order to have a uniformed distribution of the cells. Culture the cells, ON, in the 37&deg;C incubator, 5% CO2. Before infection, check the cells under the microscope to confirm a monolayer; then, proceed with the infection:
 <ul>
 <li>Wash cells, twice, with 1 ml of PBS 1X.</li>
 <li>Infect with the 200 &mu;l of centrifuged tissue culture supernatants for 1 hour at RT. Do not let the cells dry. Rock the 6-well-plate every 10 minutes.</li>
 <li>After 1 hour of viral absorption, remove the infection media from the MDCK cells and add 2 ml of DMEM 0.3%BA 1%PS containing 1 &mu;g/ml of TPCK-trypsin.</li>
 <li>At 48-72 hours after passage, depending on the transfection efficiency and the virus load, a cytopathic effect (CPE) will be observed in the MDCK infected cells. CPE suggests a successful rescue. However, an HA assay (section 2) should still be performed to confirm the presence of the virus in the tissue culture supernatants.</li>
 </ul>
 </li>
 </ol>
 </li>
 <li>Harvest allantoic fluid from infected chicken embryonated eggs: All procedures to harvest the allantoic fluid from infected eggs are performed under sterile conditions. Approximately 8-12 ml of allantoic fluid can be harvested from each 10-day-old-infected egg. Prior to harvesting the allantoic fluid, incubate the chicken eggs for 2 hours (or ON) at 4&deg;C to kill the chicken embryo and coagulate the blood.
 <ul>
 <li>Wash the eggshells with 70% ethanol to establish sterile conditions.</li>
 <li>Open the egg, carefully, over the air cavity by tapping with a spoon. Remove the broken eggshell with the help of forceps.</li>
 <li>With a 1 ml needle, remove the allantoic membrane without breaking the egg's yolk.</li>
 <li>Stabilize the chicken embryo with a spatula as you guide a 10 ml pipette into the allantoic fluid. Collect as much allantoic fluid as possible into a 15 ml centrifuge tube on ice in an ice bucket without breaking or collecting any of the egg's yolk. Use a 15 ml centrifuge tube for each egg.</li>
 <li>Centrifuge for 5 minutes at 4&deg;C and transfer the allantoic fluid (without taking pelleted red blood cells) to a fresh 15 ml centrifuge tubes.</li>
 <li>Store the tubes containing the centrifuged allantoic fluid at 4&deg;C until they are checked for the presence of rescued virus with a hemagglutination (HA) assay.</li>
 </ul>
 </li>
</ol>
2. HA assay to confirm the rescue of recombinant influenza viruses
Hemagglutination assay (HA) is routinely used to detect the presence of rescued virus in MDCK tissue culture supernatants and/or the allantoic fluid of harvested eggs. Alternatively, immunofluorescence assays (IFA) can be also performed. Once an assay identifies the presence of rescued virus, the virus should be plaque purified and the genetic composition of the virus will be confirmed by RT-PCR and sequencing.
The presence of virus in the MDCK tissue culture supernatants and/or in the allantoic fluid from infected eggs can be determined macroscopically using HA of chicken (or another source) red blood cells (RBC). The presence of virus induces hemagglutination of RBC while the absence of virus allows the formation of a red pellet in the bottom of the well (Figure 4). In the case of influenza virus, it is believed that approximately 103-104 plaque forming units (PFU) are required to give a positive signal in the HA assay; therefore, an IFA can be performed in parallel with the HA assay to confirm a true negative result. IFA with primary anti-influenza antibodies is more sensitive than the HA assay because less than 103-104 viruses can be detected with this technique. It is possible than supernatants or allantoic fluids that are HA-negative are positive by IFA. In this case, the virus should be amplified by passaging, again, in MDCK cells or in eggs. Allantoic fluid and/or tissue culture supernatants from the second passage should now be clearly positive in the HA assay.
HA assays are carried out in V-bottom 96-well plates. Negative (for instance, PBS 1X) and positive (tissue culture supernatants and/or allantoic fluid from an influenza virus infection) control samples should always be included in any HA assay to validate it.
<ul>
 <li>Dispense 50 &mu;l of PBS 1X into each well of the V-bottom 96-well plate.</li>
 <li>Add 50 &mu;l of the MDCK tissue culture supernatants and/or allantoic fluid from the infected eggs to the first well and, make 2-fold serial dilutions for the following wells. Discard the extra 50 &mu;l from the last well.</li>
 <li>Add 50 &mu;l of 0.5%-1.0% chicken red blood cells (prepared in PBS 1X) to each well.</li>
 <li>Incubate the V-bottom 96-well plate for 30-45 minutes (until a red dot is visible in the bottom of a negative control PBS sample) on ice. Read and interpretate the results as indicated in Figure 4.</li>
</ul>
3. Passage of tissue culture supernatants
A negative result in the HA assay may be a result of low transfection efficiency with low levels of virus being present in the tissue culture supernatants and/or allantoic fluid. Passage of these samples in fresh MDCK and/or embryonated eggs will allow amplification of the virus (as indicated in Figure 3) Infections are performed as previously described in section 1.11.2.
4. Representative Results
Successful influenza virus rescue will be confirmed by the presence of a positive HA assay (Figure 4). Additionally, the existence of CPE in cells infected with the tissue culture supernatants or with the allantoic fluid from eggs will suggest a positive viral rescue.
<img src="/files/ftp_upload/2057/2057fig1.jpg" alt="Figure 1" /> 
 Figure 1. Influenza Virus structure: Influenza virus is surrounded by a lipid bilayer containing the two viral glycoproteins (HA, NA) and, also, the ion channel protein, M2. HA is the viral attachment protein, responsible for binding to sialic acid-containing receptors. NA is responsible for viral release from host cells. Underneath the lipid bilayer, is a protein layer composed of the inner surface envelope matrix protein 1, M1, which plays a role in virion assembly and budding and the nuclear exporting protein (NEP), required for nuclear export of viral ribonucleocapsids. The core of the virus is made of a ribonucleoprotein (RNP) complex, composed of 8 single-stranded negative RNA viral genes encapsidated by the viral nucleoprotein, NP. Associated with the RNP complex are the viral RNA-dependent RNA polymerase subunits PA, PB1, and PB2. The non-structural proteins NS1 and PB1-F2, encoded by the RNA segments NS and PB1, respectively, are not part of the virion structure.
<img src="/files/ftp_upload/2057/2057fig2.jpg" alt="Figure 2" /> 
 Figure 2. Influenza virus rescue plasmids: The eight influenza virus genes cloned into the ambisense plasmid pDZ are indicated. pDZ plasmid 6, derived from the protein expression plasmid pCAGGs 7, is a bidirectional plasmid vector with a human RNA polymerase I promoter and a mouse terminator sequence that encodes the negative sense genomic RNA; in opposite orientation to the polymerase I unite, a polymerase II transcription cassette (chicken &beta;-actin promoter and polyA) encodes the viral proteins from the same viral gene. cDNAs from each viral segment are generated by RT-PCR with forward and reverse primers containing the SapI restriction endonuclease site and the noncoding regions of each segment (black boxes at the end of the viral genes). The PCR product is cloned into the pDZ digested with Sap-I.
<img src="/files/ftp_upload/2057/2057fig3.jpg" alt="Figure 3" /> 
 Figure 3. Eight-plasmid-based influenza rescue system: pDZ plasmids containing the 8 influenza viral genes are co-transfected, in suspension, in 293T-MDCK cells co-cultures (day 1). Twenty-four hours post-transfection, media without FBS but containing TPCK/trypsin is replaced (day 2). Forty-eight hours after changing media, tissue culture supernatant is harvested and used to infect MDCK or 10-day-old embryonated chicken eggs (day 4). 48-72 hours post-amplification, tissue culture supernatants from MDCK infected cells or allantoic fluid from eggs are harvested and assayed for presence of virus by HA (day 6). If no virus is detected, the same supernatants and/or allantoic fluids can be re-passaged into fresh MDCK cells and/or embryonated eggs.
<img src="/files/ftp_upload/2057/2057fig4.jpg" alt="Figure 4" /> 
 Figure 4. Hemagglutinin assay (HA): Hemagglutination of RBC by virus particle is visible macroscopically and is the basis to detect viral particles in tissue culture supernatants or/and allantoic fluids. Although the HA assay does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells, the assay is a good indicator of presence of virus in samples. A) Absence (top) of presence (bottom) of virus in the biological samples is determined by presence of RBC in the bottom of the plate or their absence, respectively. B) A representative result from an HA assay with no detectable levels of virus (top) or presence (bottom) of virus is shown.

<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>

Mount Sinai School of Medicine has intellectual property rights in the area of recombinant influenza viruses, and AG-S is an inventor in this intellectual property.

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 creation of, for instance, a novel restriction enzyme site is used to distinguish between wild-type and recombinant influenza virus by enzyme digestion. Therefore, after amplification of the plaque purified recombinant virus, RT-PCR and sequencing approaches should be performed to verify the nature of the rescued virus. The development of these reverse genetics techniques and successful rescue of recombinant influenza viruses from plasmids will allow you to answer specific questions about the biology of the virus.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

30.2K Views.  University of Rochester School of Medicine and Dentistry. 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.

Video: Generation of Recombinant Influenza Virus from Plasmid DNA

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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, ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...