2 methods:

1) Quick method, following clarification (2200xG, 10min @4C), pellet virus 45,000xG for 3Hr @4C or 103,000xG for 1.5Hr @ 4C.

2) If you want intact virus you can band pelleted virus from method 1 over 30%/60% sucrose step gradient (in Calcium Borate buffer) for ~1.5Hr @50,000xG 4C. Using Calcium borate buffer instead of PBS will help retain virus NA activity.

We're using a rather crude, but quick protocol for Influenza A in a mid scale (216 ml per UC run):

- spin culture s/n at 2200 g for 10 minutes

- filter s/n through 0.45 µm filters

- UC at 54 000 g (avg) for 2 hours at 4° C (in a Beckman SW32ti rotor)

- reuspend in suitable medium

I'd also recommend checking infectivity pre/post purification as well as protein amounts...

Thanks Christoph. What do you use to check protein amounts pre and post? thank you, Joyce

2 methods:

1) Quick method, following clarification (2200xG, 10min @4C), pellet virus 45,000xG for 3Hr @4C or 103,000xG for 1.5Hr @ 4C.

2) If you want intact virus you can band pelleted virus from method 1 over 30%/60% sucrose step gradient (in Calcium Borate buffer) for ~1.5Hr @50,000xG 4C. Using Calcium borate buffer instead of PBS will help retain virus NA activity.

Hi Joyce,

Any decent antibody for a structural protein would do...having said that, we haven't found any I could recommend. Therefore we're relying on measuring infectivity...

thank you Thomas and Christoph! Most helpful.

if you are looking for a highly conserved Ab for influenza there are several available to A/NP or B/NP that work very well available from Millipore (or we have some that you can borrow from CDC).

thank you Thomas! For clarification of the virus, do you use a glass wool column?

Are you referring to the gradient? Please e-mail me your contact information to [email protected] and I can speak with you about it and send you any necessary protocols.

thanks Thomas. I will shoot you an email.

This is a bit late to the discussion, but if you're still looking for protocols we've had good results with rate zonal ultracentrifugation through sucrose buffered in NTC (NaCl, Tris, CaCl2) - the details of the method are in Hutchinson et al. 2012 (PMID: 23144613). The concentration is sufficient that protein amounts post-purification can be readily assessed by silver-stain (amounts of virions can also be quickly assessed by HA assay).

This is sufficient for most applications (it's comparable in purity to vaccine-style purifications, for example) but if you require extremely pure virus an old but effective method is haemadsorption - mix clarified virus with RBCs at 4C, leave for about 15 min with occasional agitation until the HA binds the cells, rinse the cells then warm up to 37C in PBS so that the NA elutes the virus. Then purify as above.

Hope that helps!

Thanks Edward!

i am working with influenza virus recently and have been successfully amplifying influenza viruses. For ultra centrifugation i use 35000rpm for 1 hr . My ultracentrifuge id LE 80K Beckman.  These viruses are grown on MDCK.

thanks Avishekh. did you use any type of sucrose gradient to purify? 

Sorry for delayed response. No, not at all. My yield is quite high as well. 

https://www.biology.usu.edu/files/uploads/aUndergrad%20Info/Undergrad%20Research/spring_2013_undergrad_posters/Chase_Pease_-_Poster.pdf

It would be helpful. Although it is not written for influenza virus that propagated in eggs purification, we apply the condition and it works fine.

Feng, I sent you a protocol for the purification from eggs. Sorry for the delay. I hope it works! Good luck, Joyce 

Hi Avishekh

I know its been a while since your post but can I ask how much culture supernatant from infected MDCK cells you use and do you perform any centrifugation steps to remove cell debrigh before ultracentrifuaction.

I am only using 2ml of supernatant, I`m now guessing this is far too little?. I was also advised to spin at 100,000g for just 20 minutes, is this too short?

There are so many different lengthy protocols out there and I want the simplest.  I am spinning beta-propriolactone (BPL) treated viral supernatant I want to use the virus for the haemagglutination inhibition assay. I am not getting any virus detection (haemagglutination) in the assay.

At the moment I am not inactivating the BPL  so I`m worried there is carry over into the assay from the residual liquid thats remains close to the pellet (and cannt be removed because this will probably disturb the pellet) and is therefore in the resuspendant

Thanks for your help

Mark Quinlivan

Mark, the 2ml sounds too small a volume to me. I have not worked with BPL inactivated virus, however with non-BPL inactivated virus, I use all of the supernatant and clarify at low speed (~300g), 4 deg, for about 15 mins. I then further purify the supernatant at high speed (40,000g) for 2 hours, re-suspend the pellet in calcium borate and leave the tubes overnight at 4 deg to allow any residual  virus to come off the tube. I use about 4ml of buffer for each T-175 that I harvest. Our centrifuge did mess up once and it only went for 45 mins and we still got a pretty good yield, so 2 hours might be overkill. 

HI Joyce,

Could you please send me the protocol for the purification from eggs.

Thanks

I have a sneaking suspicion that BPL may interfere with HA assay - I haven't tried this, but it seems likely to interfere with sialic acid binding.

As for influenza purification protocols, we recently published this (optimised on MDCK but also works for eggs):

http://www.nature.com/protocolexchange/protocols/3315

and a wide range of protocols are reviewed at this excellent online resource:

http://www.axis-shield-density-gradient-media.com/virusindexes.htm

Hope that helps,

Ed

We BPL treat all viruses which we include in the influenza WHO kit for use for influenza surveillance and have found to interference with HA binding.

Thanks Thomas - that's good to know!

Sorry, meant to say we have found "no" intereference with BPL treated viruses

 Sure, no problem, Mohamed. Do you have a contact email that I may send to? 

Oh, sorry, Ed sent you some nice links. Didn't see those! 

Thanks Joyce. It is [email protected]

Do you inactivate the virus after purification?

Mohamed, just sent you an email. So sorry for the delayed response...good luck! 

Thanks Joyce for your help. Unfortunately i didn't receive the email, could you please send it again.

Thanks again for your cooperation

I re-sent to [email protected]. If you don't get it, send me another email address. :) 

Thanks i got it. I really appreciate your prompt response.

Hello Joyce,

    Could you please send me ([email protected]) the protocol for the purification from eggs? I will be very grateful for that.

I don't want to cut in on Joyce's answer, but you may find this protocols paper useful as well: Eisfeld, Neumann and Kawaoka (2014) Nature Protocols 9 2663-2681:

http://www.nature.com/nprot/journal/v9/n11/full/nprot.2014.180.html

Thank You Edward for Your help

Alicja, I sent you a protocol for the propagation and purification and also a link to another topic similar to yours. Hope it helps and best of luck!! 

Not a bother Edward! The more science, the better :D 

Thank You Joyce!

  Hello~ Joyce,

    Could you please send me the protocol for the purification from eggs?

   my email is [email protected]

   I am struggling with that problem...

   and is there are a way to purify small volume of samples (less than 2ml)?

  many thanks to you

Sure, no problem, Sang!  I will message you . 

Hello Edward,

Thanks a lot for your protocol ! I was wondering about why using NTC buffer in particular instead of others ( like NaCl Tris or NTE : NaCl Tris EDTA..). Did you determine a beneficial effect of Ca2+ on influenza virus stability during purification ?

Thank you ! Laurent

Hi Laurent,

I'm afraid it was much more a case of 'whatever works' than a systematic optimisation. I was trying to replicate a protocol with NTE (which others have successfully used) but was struggling with it. I then switched to an alternative protocol which used NTC and things started working. The buffer might have been the key, but many other things were changed at the same time. Sorry not to be more use. I suspect that the osmolarity and pH of the solution are the main factors, but I haven't tested this.

One thing which probably does make a difference is the density medium - as you probably know, sucrose gradients tend to deform particles more than isotonic media such as OptiPrep (iodixanol). Depending on your downstream application, you might also consider pre-fixation of virus particles (see Sugita et al. (2011) JGV PMID: 21795472).

Sorry not to be more of a help. Best wishes,

Ed

I believe that the Ca2+ in the Calcium borate was beneficial for retaining influenza enzymatic activity (I think for NA).

I believe that is correct, Thomas. The Ca2+ is required by the NA for catalytic activity. 

Thank you Thomas and Joyce, i had not seen your previous answer in the discussion thread Thomas !

And thank you Edward, your explanations are very helpfull ! By digging a little bit, it seems that the hyperosmotic stress due to the high sucrose concentrations could be in fact partly responsible (added to centrifugal force) for the membrane alterations observed. I rapidly calculated that 20 and 60% sucrose-NTE or sucrose-NTC (with usual buffer composition) should have an osmolarity of respectively ~0,85 and ~2 Osm, while classical cell culture media and viral cytoplasm are ~ 300 mOsm. In the source, they characterized the viral shrinkage and decrease in HA activity associated with hypertonic osmotic difference across viral membrane (see Choi et al. (2015) http://dx.doi.org/10.1371/journal.pone.0134431). This can be related to morphologic alterations pointed out by Sugita et al. after a 20% Sucrose UC, and justifies utilization of iodixanol.

The pH of the solution should also play a role because it surely will influence particle aggregation...(I know that has been tested for influenza VLP : Kissmann et al.(2010) DOI 10.1002/jps.22304 but did not find anything for influenza...)

Thomas and Joyce's point about NA activity and Ca2+ is really useful and something I'd forgotten about. Another issue with pH is of course the premature activation of HA at low pH (typically

Hi Joyce Sweeney. I see it had been a year since you had discussion about purification of influenza virus but do you remember on top of your head how much virus were you able to purify after ultracentrifugation of your clarified cell lysate?

Hi Muddassar, for the titer I believe I got between 10^6 and 10^8, depending on the strain I was concentrating. Volume wise I used a ml of PBS to resuspend each pellet. Does that answer your question? Message me if you need further help!

Good luck,

Joyce

That helps. Comparatively do you think sucrose gradient ultracentrifugation would give better yield? Still this is good enough though!

Well, the gradient will remove more impurities. I haven't done a side by side comparison of the two so I am not sure how it would affect yield. I don't think there would be much difference in the yield but I haven't done a side by side comparison. :-)

Hi, Joyce

would you give me the protocol purifying influenza virus from egg contents?

My e-mail is [email protected]

Thanks.

Hi there,

Anyone knows what is the rough yield estimate of zonal centrifugation for HA purification in industrial scale? I have heard that the maximum loading volume of zonal centrifugation is 2 L. However our fermentation volume may goes up to 2000L.

Thanks.