Purification of Virus from the Allantoic Fluid of Embryonated Chicken Eggs

Begin with a reservoir containing allantoic fluid harvested from virus-infected chicken eggs.

This fluid contains virus particles along with host-derived contaminants.

Pass the fluid through a depth filter to capture large contaminants while allowing smaller contaminants and viruses to pass through.

Then, load the filtered fluid into a filtration system, where it flows tangentially across a membrane surface.

Smaller contaminants pass through the membrane, while the virus is retained and concentrated.

Load the concentrated virus onto a density gradient and centrifuge it. The virus forms a distinct band where its density matches the gradient.

Collect the virus-rich band in a dialysis cassette and place it in a buffer. The gradient components diffuse out, while water from the buffer diffuses in through the semipermeable membrane.

Next, place the cassette in a hypertonic solution to draw out water and concentrate the purified virus.

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 apical 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 conformation 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 elution, 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 waste lines.

Uncouple the retentate 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 retentate 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 ultracentrifuge 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 eluted 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.