1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine
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Martínez-Sobrido, L., García-Sastre, A. Generation of Recombinant Influenza Virus from Plasmid DNA. J. Vis. Exp. (42), e2057, doi:10.3791/2057 (2010).
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.
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.
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.
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.
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.
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.
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.
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.
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.
The authors want to thank past and present members in the Adolfo García-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.
|DMEM||Invitrogen||11995-065||Store at 4°C|
|OptiMEM||Invitrogen||51985-034||Store at 4°C|
|Lipofectamine 2000 (LPF2000)||Invitrogen||11668-019||Store at 4°C|
|TPCK-trypsin||Sigma-Aldrich||T-8802||Store at -20°C|
|Bovine Albumin (BA)||Sigma-Aldrich||A7979||Store at 4°C|
|Trypsin-EDTA||Invitrogen||25300-054||Store at -20°C|
|Penicillin/Streptomycin (PS) 100X||Invitrogen||15140-122||Store at -20°C|
|Fetal Bovine Serum (FBS)||Hyclone||SH30070.03||Store at -20°C|
|V-bottom 96-weel plates||Nalge Nunc international||249570|
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°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°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°C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80°C.
All plasmids are prepared using a plasmid maxi kit following manufacturer’s recommendations. All plasmids are aliquot at concentrations of 1 μg/ml in ddH2O and stored at -20°C. For short-term storage, the plasmid can be keep at 4°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.
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°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°C. Just before use, add TPCK treated trypsin to a final concentration of 1 μg/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.