The overall goal of this procedure is to demonstrate a simple and highly reproducible protocol for the generation of recombinant arena viruses in FDA approved Vero cells for potential vaccine or vaccine vector seed development. This is accomplished by first carefully preparing the plasmid DNA transfection mixture. The second step is to transfect Vero cells with the plasmid DNA transfection mixture.
Next, it is necessary to scale up transfected cells into 100 millimeter dishes. Ultimately, the final step is to confirm a successful virus rescue by immunofluorescence using wild type virus or bi fluorescence microscopy and GIA luciferase expression using tris segmented virus Compared to existing methods such as rescues and rine cell lines. This protocol generate renal viruses in Vero cells, which are approved by the FDA for the development of vaccines.
In addition to the generation of potential vaccine seed, the reported gene expressing tri segmented virus can also be applied for the identification of antivirals against VIRs in head throughput screens without the need of secondary assays for viral detection. First, prepare 250 microliters of optimum media and depending on the virus, rescue 10 to 12 micrograms of lipo 2000 per transfection incubate for five to 10 minutes at room temperature. During this time, prepare the plasma transfection mixture in a separate micro centrifuge tube using the recommended amounts for each virus rescue.
Bring the final volume to 50 microliters with optimum. Next at 250 microliters of the optimum lipo mixture into the plasma DNA transfection mixture and incubate for 20 minutes at room temperature. To prepare Vero cells first equilibrate, the P-B-S-D-M-E-M 10%FBS 1%PS media and the trypsin EDTA mixture to 37 degrees Celsius to harvest the bureau of cells, wash the cultures twice with five milliliters of PBS.
Then add one milliliter of trypsin EDTA for cell detachment if necessary, gently tap to fully detach the cells. Now re suspend the cells in 10 milliliters of complete DMEM in a 15 milliliter centrifuge tube. Then centrifuge the cells for five minutes at 1000 RPM re suspend the cells in 10 milliliters of complete DMEM.
Count the cells and adjust concentration to one times 10 to the six cells per milliliter. After the 20 to 30 minutes room temperature incubation, add one milliliter of Vero cells to the optimum lemine, DNA plasmid mixture and incubate for five minutes at room temperature. Now seed the lipo DNA cell mix into six well plates.
Gently shake and then place in the incubator. After 16 to 24 hours, post transfection, remove the transfection media and add two milliliters of infection media culture, the cells for an additional 48 hours. Next to passage, the cells remove the tissue culture supernatant wash twice with one XPBS and trypsin ice the cells as shown earlier.
Then resuspend the cells in one milliliter of complete DMEM to pellet the cells centrifuge for five minutes. At 5, 000 RPMs and four degrees Celsius. Carefully resuspend the pellets in one milliliter of infection media and transfer the cells to a 100 millimeter dish.
With infectious media, bring up the total volume in the plate to eight milliliters, a sufficient volume to prevent drying of the cells, as well as for concentrating the virus. Gently shake the 100 millimeter dish for a uniform monolayer of transfected cells after culture. After 72 hours, collect the tissue culture supernatants centrifuge to remove the cell debris and store the supernatants at minus 80 degrees Celsius the day before titration.
Wash the Vero cells twice with PBS and trypsin ice. After resus suspending the cells in 10 milliliters of complete DMEM. Count the cells with a hemo cytometer and adjust the concentration to four times 10 of the four cells per well then prepare 96 well plates to reach 80 to 90%co fluency.
The next day. Gently shake the plates by hand to get a uniformed distribution of the cells. Culture the cells for overnight on the day of infection.
Check the cells under the microscope to confirm a monolayer before proceeding with the infection. Now serially dilute the virus containing tissue culture supernatants that were recovered earlier in optimum in a 96 well plate then aspirate the media from the seeded Vero cells. Wash twice with 50 microliters of PBS and infect cells with 50 microliters of the serially diluted virus.
Infect the cells for one and a half hours at 37 degrees Celsius and 5%carbon dioxide. Next, remove the virus inoculum at 100 microliters of infection media per well and incubate the cells for 16 to 18 hours. Now remove the tissue culture supernatants from each.
Well fix the cells with 4%formaldehyde diluted in PBS for 15 minutes at room temperature. Then remove the formaldehyde and permease with 0.1%trite X 100 at room temperature for 10 minutes. Next, aspirate the permeation solution and wash three times with 100 microliters of PBS lock cells with 2.5%bovine albumin serum.
Dilute the primary antibody specific to an arenavirus protein in 2.5%BSA and centrifuge for 15 minutes. At 3, 500 RPM, incubate the cells with the antibody for one hour at 37 degrees Celsius after three PBS washes at an appropriate fluoro four conjugated secondary antibody for 30 minutes at 37 degrees Celsius. Remove the secondary antibody and wash three times with 100 microliters of PBS.
Next titrate by counting fluorescent focus forming units using fluorescence microscopy. Similar to wild type recombinant arena virus. Titrate the tric segmented virus by first serial diluting the virus present in the tissue culture, supernatant then infect Vero cells in 96, well plates after 16 to 18 hours of incubation titrate by counting fluorescent focus, forming units using fluorescence microscopy.
Alternatively, measure successful virus rescue by Gaia luciferase expression using 100 microliters of tissue culture supernatants in an 96 well white plate. Set up the luminometer as per the manufacturer's recommendations. Add 50 to 100 microliters of Gaia assay solution to each sample.
Then measure the Gaia reporter gene expression with the luminometer as a negative control. Measure Gaia expression from TCS of wild type virus infected cells. Arena viruses are enveloped negative sense RNA viruses with bi segmented genomes.
Each segment uses an ambi sense coating strategy to direct the synthesis of two viral proteins. In opposite orientation, two types of rescue plasmids are used for the generation of recombinant arenavirus in Vero cells. PCA's expression plasmid direct the synthesis of the viral proteins L and np.
The human polymerase one plasmid directs the synthesis of the viral RNA segments. Successful rescue of a recombinant wildtype arena virus is confirmed by the presence of viral antigens using IFA at 16 to 18 hours. Post-infection of Vero cells by tissue culture, supernatants cells are stained with LCMV or canid number one specific antibodies.
In tris segmented arenavirus rescues the human polymerase one plasmid is separated into two plasmids in one plasmid. NP is replaced with GFP and in the other plasmid, the GP is replaced by Gaia gene. Successful viral rescue can be simply assessed by observing GFP expression by fluorescence microscopy.
Furthermore, successful rescue can be confirmed by assessing Gaia expression. After watching this video, you should have a good understanding of how to rescue both wild type and tris segmented arena viruses in Vero cells with high reproducibility. We recommend three independent transfect for each recommended virus to increase the likelihood of our successful rescue as severe cells are not easily transfected.
