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 μl of OptiMEM media and 6-8 μl 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 μl of OptiMEM media. We usually use 1 μg of each influenza DNA plasmid per rescue. Add 1 μl of the pDZ plasmids (at 1 μg/μl) PB2, PB1, PA, HA, NP, NA, M, and NS to a tube containing 50 μl of OptiMEM media.</li>
 <li>OptiMEM-LPF2000-DNA plasmid mixture: Add 250 μl 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°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 μl 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 μl 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 μl 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°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 μg/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 μl of centrifuged tissue culture supernatants from step 1.10. Incubate the cells and/or eggs at 37°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 μl 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 37οC 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°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 μl 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 μg/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°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°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°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 μl of PBS 1X into each well of the V-bottom 96-well plate.</li>
 <li>Add 50 μl 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 μl from the last well.</li>
 <li>Add 50 μl 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="Influenza virus structure diagram; labeled proteins HA, NA, NP, M1, M2; virology concept." data-altupdatedat="1747911167000" data-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="Genetic cloning diagram, DNA insertion into pDZ plasmid, viral genes, and promoters annotated." data-altupdatedat="1747911167000" data-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 β-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="Plasmid transfection diagram, 293T/MDCK co-culture, HA assay, virus passage in MDCK/eggs." data-altupdatedat="1747911167000" data-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="Virus rescue schematic: diagram of virion impact with dilution series; experiment result comparison." data-altupdatedat="1747911167000" data-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.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11995-065</td><td>Store at 4&amp;#x00B0;C</td></tr><tr><td>OptiMEM</td><td>Invitrogen</td><td>51985-034</td><td>Store at 4&amp;#x00B0;C</td></tr><tr><td>Lipofectamine 2000 (LPF2000)</td><td>Invitrogen</td><td>11668-019</td><td>Store at 4&amp;#x00B0;C</td></tr><tr><td>TPCK-trypsin</td><td>Sigma-Aldrich</td><td>T-8802</td><td>Store at -20&amp;#x00B0;C</td></tr><tr><td>Bovine Albumin (BA)</td><td>Sigma-Aldrich</td><td>A7979</td><td>Store at 4&amp;#x00B0;C</td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td>Store at -20&amp;#x00B0;C</td></tr><tr><td>Penicillin/Streptomycin (PS) 100X</td><td>Invitrogen</td><td>15140-122</td><td>Store at -20&amp;#x00B0;C</td></tr><tr><td>Fetal Bovine Serum (FBS) </td><td>Hyclone</td><td>SH30070.03</td><td>Store at -20&amp;#x00B0;C</td></tr><tr><td>V-bottom 96-weel plates</td><td>Nalge Nunc international</td><td>249570</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Embryonated chicken eggs&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;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&amp;#x00B0;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.&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Chicken red blood cells (RBC)&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;Chicken RBC can be purchased from Truslow Farms, 201 Valley Road, Chestertown, Md 21620. Store at 4&amp;#x00B0;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).&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Tissue culture supernatants and allantoic fluids&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;Both, tissue culture supernatants and allantoic fluids can be stored at 4&amp;#x00B0;C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80&amp;#x00B0;C.&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Plasmids&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;All plasmids are prepared using a plasmid maxi kit following manufacturer&amp;rsquo;s recommendations. All plasmids are aliquot at concentrations of 1 &amp;#x03BC;g/ml in ddH&lt;sub&gt;2&lt;/sub&gt;O and stored at -20&amp;#x00B0;C. For short-term storage, the plasmid can be keep at 4&amp;#x00B0;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.&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Viruses&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;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.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;Tissue culture media and solutions&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;DMEM 10%FBS 1%PS: 445 ml Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4&amp;#x00B0;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&amp;#x00B0;C. Just before use, add TPCK treated trypsin to a final concentration of 1 &amp;#x03BC;g/ml. Infectious media.&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;10X Phosphate buffered saline (PBS): 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;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;1X PBS: Dilute 10X PBS 1:10 with ddH&lt;sub&gt;2&lt;/sub&gt;O. Sterilize by autoclave and store at room temperature.&lt;/p&gt;</td></tr></tbody></table>

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

30.4K Views.  University of Rochester School of Medicine and Dentistry. Redding van influenza A-virussen uit het plasmide DNA is een fundamentele en essentiële experimentele techniek die het mogelijk maakt influenza onderzoekers recombinante virussen te genereren om meerdere aspecten in de biologie van influenza virus te bestuderen en om te worden gebruikt als potentiële vectoren of vaccins.

Video: Generation of Recombinant Influenza Virus from Plasmid DNA

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 ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

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 Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

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 ...

Biology

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Source: Arevalo, M. T., et al.&#160; <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54041">Expression and Purification of Virus-like Particles for Vaccination.</a> J. Vis. Exp. (2016).
This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Source: Arevalo, M. T., et al.&#160; Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).
This video demonstrates the ...

Generation of Recombinant Influenza Virus from Plasmid DNA

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