It might be that the fluid is at a similar density to the virus, meaning that it acts as a cushion. I assume that you are using something like HBSS or similar to make the volume back up? This will decrease the density of the allantoic fluid, and enable you to spin the virus out of suspension.

Please see attached article. Should help

Here is the article

Thank you Matt & John. To purify a smaller amount of virus I could load the allantoic fluid directly onto a gradient as in the paper mentioned by John. To purify larger amounts the volume of allantoic fluid is such that we routinely pellet the virus before layering it onto a gradient. If you are right Matt, I could simply dilute the allantoic fluid (maybe 1:1) in PBS & see if I get better initial recovery, then move onto the sucrose gradient.

The allantoic fluid may also contain viruses of differing morphologies and/or densities such that only certain viruses pellet.

I think that the morphology could definitely be important. I am not sure if filamentous particles are more easily lost. I was just curious, are you doing a low speed spin or filtering to clarify your allantoic fluid before ultra centrifugation? What method are you using for comparing virus particles before and after (HA, plaque, TCID)?

Also, I have done a 1:1 dilution in PBS before spinning and used a 20% cushion, but I do not really have a comparison to know if it is better than the method you already tried.

James - I do a low speed spin to clarify the allantoic fluid before ultracentrifugation. I am using a virus counter (from ViroCyt) because it is quicker than plaque & more accurate than HA. HA gives the same result.

I have also tried using PEG and a low speed spin to precipitate virus from allantoic fluid & although I have less experience there, it seems like the 1:1 dilution in PBS improves yield. This might be my new approach for large volumes of allantoic fluid.

Yes, I had purified orthomixo and parmayxovirus, and the first step with low speed centrifugation with diluted allantoic fluid (1:1 or more diluted) improves virus recovery.

We routinely pellet influenza viruses by ultracentrifugation. As above, we clarify the allantoic fluid before ultracentrifugation. What G forces are you using? For large volumes, we pellet the allantoic fluid directly, without diluting @ ~45,000 x G for 3Hr. For purification, we then next band the virus over a 30/60% Sucrose (~105,000 x G, 1.5Hr).

Using an SW40 rotor I calculate that i am pelleting the larger volume @~67,000 x G for 1.5 hours. Maybe I need a longer spin time here. The purification by banding the virus at 30/60 sucrose gets almost complete recovery at @~ 45,000 x G for 1.5 hours.

Hi

We do a 5 step centrifugation with concentration for viruses reproduced in chicken embryos.

1. 2 000 g 30 min

2. 5 000 g 30 min

3-5. 35 000 g 30 min per step (virus precipitates at this stage; first step here is concentration ~1 to 10; then concentrated suspension is purified 2 more times under same conditions)

PBS is used for re-suspending at all steps.

Usually ~10-15 ml are extracted per embryon and our virus lot is ~100 embryos. So we use 6*250 ml for high speed centrifugation and then 6*~40 ml for ultracentrifugation. We also prefer bucket rotors than angled (when available).

The concentrated and purified virus shows stable HA, EID (embryon infectious dose) and TCD (MDCK) titers even when stored for 6 months at 4 C.

These suspensions do not react in immune-diffusion and immune electrophoresis against sera from rabbits immunized with allantoic fluid from non-infected embryos (sera from rabbits immunized with vaccines based on the respective strains have high titers against these viruses).

We use this approach for experimental adjuvanted influenza vaccine preparation as well as immunogenicity and efficacy/effectiveness evaluation of the vaccines. Vaccines based on purified and concentrated suspensions are significantly more immunogenic (without adjuvants: vaccine + PBS) in mice than those with the addition of purified allanotic fluid (without adjuvants: vaccine + allantoic fluid).

For other uses further purification (surcose or even cesium chloride gradient) may be necessary.

Regards

Yuri Vasiliev gave a very detailed protocles for pelleting influenza viruses with alloantoic fluid. We use simmilar methods, Usually we used at least 90 eggs for a around of purification. First, 3000rpm, 15 minites to remove some floppies from the fluid, then pellet the virus with 6000rpm overnight. Afterwards, resupsend the virus pellet with 2-3ml of the supernant, mix thoughly and load on the sucrose gradient after disrupt the pellt.