The overall goal of this procedure is to rescue recombinant Newcastle disease virus or NDV from cloned DNA. This is accomplished by first infecting mammalian cells with a helper virus encoding T seven polymerase, and then transecting those cells with plasmids, encoding the minimal replication machinery of the virus under control of the T seven promoter. The second step is to co cultivate the transfected mammalian cells with avian cells, which are from the natural hosts of the virus.
The newly generated varians released from the mammalian cells will infect the avian cells for a first round of amplification. Next, the supernatant of the co-culture is inoculated into embryonated chicken eggs for further growth of the rescued virus, the final step is to collect the LAN toic fluid from the inoculated eggs. Ultimately, a simple Hemagglutinin nation assay is used to show the presence of NDV in the LAN toic fluid and the success of the rescue process.
Recombinant viruses rescued by this method can provide insights into different factors affecting infection and can also be used as vectors for the delivery of antigens for vaccination and antitumoral therapy. To begin split mammalian hep two or a 5 49 cells the day before transfection ins six well plates, the density of the cells should reach 80 to 90%confluence the following day. For each virus to be rescued, include two to four different wells.
Also include two extra wells for the controls PCAs, GFP and pite, GFP aimed to monitor transfection and MVAT seven infection efficiencies respectively. Wash cells twice with one milliliter of warmed PBS, then infect the cultured mammalian cells with the MVA T seven virus at a multiplicity of infection of one PFU per cell. In a final volume of 200 microliters in PBS, leave the cells to incubate for one hour at room temperature during the viral infection incubation, prepare the transfection mix as described in the written text after the lipectomy optimum solution has been prepared and pre incubated for five minutes.
A 250 microliters of the solution to the DNA tubes and incubate for 20 to 30 minutes at room temperature following incubation with MVAT seven, remove the virus inoculum by aspiration and replace with the DNA lipo transfection mix. Add one milliliter of DMEM 10%FBS to each dish. Incubate the infected transfected cells for six to eight hours at 37 degrees Celsius, 5%carbon dioxide.
Then replace the transfection medium with 1.5 milliliters of fresh DMEM 10%FBS At 24 hours post transfection control wells can be observed under a fluorescence microscope to assess transfection efficiency and T seven polymerase activity before proceeding with co-culture of the infected transfected mammalian cells with avian fibroblasts. Usually a confluent 100 millimeter tissue cultured dish of chicken or duck embryo fibroblasts is used per two transfected wells. Be sure to prepare in advance enough.
100 millimeter tissue culture dishes of avian cells per all rescue attempts for efficient rescue of the virus. DMEM 10%FBS medium is supplemented with 5%elan toic fluid and 30 millimolar magnesium chloride. After washing the mammalian cells twice with warmed one XPBS trypsin eyes the cells with 0.2 milliliters of EDTA trypsin until they detach.
Re suspend the cells in one milliliter of the supplemented DMEM and transfer to a 100 millimeter tissue culture dish at three milliliters of the same medium. After washing the avian cells twice with warmed one XPBS trypsin eyes the cells with one milliliter of EDTA trypsin and incubate for one to two minutes at 37 degrees Celsius until cells detach. Re suspend the avian cells by adding eight milliliters of the prepared medium.
Then add four milliliters of the tryps inized avian cells to the mammalian cells in the co-culture 100 millimeter tissue culture dish For a total volume of eight milliliters. Gently shake the co cultured cells in the 100 millimeter dish to ensure a uniform distribution and then place them in the incubator at 37 degrees Celsius for three to four days. Eggs are infected when all of the co cultured cells are affected by the cytopathic effect due to the MVAT seven viral infection.
At this point, remove one milliliter of the tissue culture supernatant and add it to an eend DPH tube centrifuge. The tissue culture supernatant for one minute at 12, 000 RPM in a benchtop centrifuge. To remove cellular debris, candle the eggs using a light candling box to see the interface between the air sac and the Alan Toic cavity.
Make a mark on the interface border avoiding blood vessel localization. Spray 70%ethanol over the eggs to establish sterile conditions. Gently make a hole in the eggshell on the marked spot.
Then inoculate 500 microliters of the snat using a one milliliter syringe, aiming the needle vertically and directly into the Alan Toic cavity. Seal the nick in the eggshell with melted wax or paraffin. Incubate the eggs for two to three days At 37 degrees Celsius.
Incubate the infected eggs for two to four hours or overnight at four degrees Celsius in order to kill the chicken embryo and coagulate the blood. Spray the eggs with 70%ethanol to maintain sterile conditions. Carefully tap the apical section of the egg over the air cavity with a spoon.
Once the egg eggshell is cracked, remove the fragments with forceps and fully expose the LAN toic membrane. Expose the LAN toic cavity by excising the ENT toic membrane with forceps and scissors, avoiding damage to the blood vessels and the oak. Carefully push down the embryo with a spatula and collect the up flowing LAN toic fluid with a 10 milliliter pipette.
Avoid damaging the yolk membrane. Transfer the LAN toic fluid to a 15 milliliter centrifuge tube on ice. Clarify the LAN toic fluid by centrifuging for five minutes at 1500 RPM.
Transfer the supernatant to a fresh tube without disturbing the pellet. Store the samples at four degrees Celsius for up to one week until checking them for the presence of NDV by the Hemagglutinin Nation assay. The presence of virus in the Alan Toic fluid from infected eggs can be determined macroscopically by their ability to hemagglutinin eight Turkey red blood cells negative control samples such as one XPBS or uninfected Alan Toic fluid as well as positive control samples such as Al Toic fluid from any NDV virus should always be included in any HA assay to validate it.
To perform the HA assay pipette 50 microliters of one XPBS per well in a V bottom 96 well plate then pipette 50 microliters of the LAN toic fluid samples into the wells on the first column of the plate. Perform twofold serial dilution through the rest of the plate and discard the last extra 50 microliters from the wells. In the last column, dispense 50 microliters of 0.5%Turkey red blood cells in one XPBS per well gently shake the plate by tapping incubate the plate at four degrees Celsius for 30 to 45 minutes or until a clear pellet is formed in the negative control wells.
Early steps in the NDV rescue procedure can be monitored, specifically the transfection efficiency and the infection with MVAT seven shown here. A representative fluorescent and brightfield images of a 5 49 cells transfected with PAGs GFP or infected with MVAT seven and transfected with P eight GFP constitutive. GFP expression driven by PCAs is an indicator of transfection efficiency, whereas T seven promoter driven GFP expression by P site is a control for MVAT seven infection and T seven polymerase activity.
The images show transfection and transfection infection efficiencies that are enough for a successful NDV rescue after the rounds of amplification in avian mammalian co cultures and embryonated eggs, presence of rescued virus is detected by positive wells in the HA assay. NDV induces hemagglutinin by linking erythrocytes and thus prevents their sedimentation in the bottom of the V-shaped well. Therefore, lack of HA activity can easily be distinguished by the formation of a red round pellet of red blood cells as shown here in the negative control and one negative result from three unknown samples obtained after rescue Rescue.
After watching this video, you should have a good understanding of how to rescue recombinant Newcastle disease virus from CDNA with high reproducibility.
