This protocol outlines the techniques used to produce Newcastle Disease Virus so that it can be administered safely to mice and other animals such as hamsters and sheep. The use of tangential flow filtration allows for large starting volumes to be processed more efficiently compared to existing procedures that use only centrifugation processes. This is a lengthy procedure involving several intricate steps, meaning time management may be an issue.
I would recommend identifying all potential pause steps so that the technique can be split up appropriately, minimizing stress. Demonstrating this procedure will be Alex, a research assistant from my laboratory. For the purification of Newcastle Disease Virus or NDV, first, prime the previously set experimental system by running 50 milliliters of PBS through it.
Stop the pump when approximately five milliliters of PBS are left in the tube. Next, attach a depth filter with a one to three micromolar retention rating to the tubing. To vent the filter, remove the second cap on the epical side of the depth filter then start running an additional 50 milliliters of PBS through the system.
Once the PBS begins to flow through the vent at the top of the filter, close the port and continue to flow PBS through the lines. After that replace the waste vessel with a new sterile collection vessel and begin to run Allantoic Fluid through the depth filter. Next, set up the tangential flow filtration or TFF system in an open confirmation and run as described in the manuscript.
When there are about five milliliters of fluid left in the reservoir, pause the pump and add depth filtered Allantoic Fluid to the reservoir. Then resume the pump flow. To prevent shearing of the virus, it's important to ensure that the pressure of the system does not exceed 10 pound force per square inch.
To increase the illusion, a combination of the speed of the peristaltic pump and C-clamp should be used. 150 to 100 milliliters of Allantoic Fluid are left in the reservoir, pause the pump and exchange the buffer by adding 150 to 200 milliliters of ML buffer. Again, pause the pump when five to 10 milliliters of fluid are left in the reservoir, then using two C-clamps close the waistline.
Uncouple the retented line feeding the reservoir and insert it into a 50 milliliter conical tube. Then resume the flow and pause when there are few drops of fluid left in the reservoir tank. Next, remove the C-clamps from the waste lines and reattach the retented feed line to the reservoir tank.
Meanwhile, to prepare for iodixanol density gradient ultracentrifugation, first add a 0.5 milliliters of 40%iodixanol to a 13.2 milliliter open top thin wall ultra centrifuge tube. Then make the column ready with successive additions of 2.5 milliliters of 20%iodixanol, and 2.5 milliliters of 10%iodixanol. Before ultracentrifugation, carefully add six to 6.5 milliliters of the virus solution alluded from the TFF system to the iodixanol column without disturbing the gradient.
After ultracentrifugation, remove the tubes with a pair of sterile forceps. The target band should be a large band between the 10 and 20%gradients. Next, suspend the tube over the top of a beaker using a retort stand.
After that, attach a 1.5 inch needle of 18 gauge to a five milliliter syringe then puncture the side of the ultracentrifuge tube with the needle and remove the target band by slowly extending the plunger. After removing iodixanol from the virus solution as described in the manuscript, use a syringe to carefully inject the virus into a dialysis cassette. To concentrate the virus solution, remove the dialysis cassette from the dialysis buffer and place it in a small sealable plastic bag.
Then add a 15 to 25 milliliters of 40%polyethylene glycol to the bag so that the dialysis cassette is completely submerged. After dialysis briefly rinse the dialysis cassette with PBS. Next, fill a syringe fitted with a 1.5 inch needle of 18 gauge with air, insert the syringe into the dialysis cassette and push the plunger.
Rotate the apparatus for removing the virus without removing any of the introduced air. To dislodge residual virus adhering to the membrane, carefully massage the membrane with gloved fingers without damaging it. To ease the dislodging process, remove some of the excess air in the dialysis cassette.
For quality control assays, first, dilute the virus solution 100 times by adding 10 microliters of it to 990 microliters of PBS in a 1.5 milliliter tube. Then continue serial dilution by adding 100 microliters of the previous dilution to 900 microliters of PBS. For infection assays, add 20 microliters of each delusion to each well of a 96 well plate.
Before examining the cytopathic effect of green fluorescence protein, or performing an indirect immunofluorescence assay, incubate the plate at 37 degrees Celsius with 5%carbon dioxide for at least 48 hours. By using this protocol, highly purified NDV was obtained as demonstrated by sodium do sulfate polyacrylamide gel electrophoresis of the viral proteins. The infectivity of the virus purified by this method was also verified by the 50%tissue culture infection dose or TCID50 assay done in 96 well plates.
The TCID50 was determined from the number of positive wells for each dilution by using the Spearman-Karber calculator. The infection assay revealed the difference between the cytopathic effects of lentogenic and mesogenic NDV on DF one cells under different conditions after 10 and 24 hours of incubation. The immuno florence assay was also effective in studying the infection of Vero cells by lentogenic NDV.
When priming the experimental system, ensure that the lines are primed effectively as any residual sodium hydroxide will inactivate the virus. Also is important to maintain membrane integrity as compromising this will significantly impair virus purification and yield.
