What protocol are you using? Farhad, ViraSource

What is your current protocol? Farhad, ViraSource

Hi, Marie-Claude,

I think, that the High Pure Viral Nucleic Acid Kit of Roche will do a good job for you.

Sincerly,

Andreas

I suggest something like that: http://www.sciencedirect.com/science/article/pii/S0166093412002571

I use iodixanol gradient purification (also called OptiPrep) for HSV-1 (also an enveloped virus) and I expect that a similar protocol would work for you. Their protocols are listed here: http://www.axis-shield-density-gradient-media.com/virusindexes.htm

For HIV, I generally do a sucrose cushion 20% spin (100000g for 2h00 at 4 deg C -- density gradient) then I would do an Optiprep.

Good luck.

For HCV, enveloped virus, purification, I used iodixanol gradient as suggested by Katherine Davoli, but then you need to find the best fraction. You could test that through WB directly and detect with specific antibodies.

I think that for high level purification of a rather fragile virus, cesium-chloride gradient and ultracentrifugation remains the "gold standard" method.

I agree with the above comment. pelletting the virus through a sucrose pad is not good for EM, you need to band the virus through a gradient.

also, you may want to consider replacing growth media with serum-free media later in the infection.

As far as I've seen all protocolls so far make use of ultracentrifugation. Which is quite the obvious solution, density cushion or not. If you haven't got access to an UC or your virus is especially brittle (hard to imagine with anything associated with archaea) we've had good experience with ultrafiltration using Sartorius Stedim columns (MWCO 1 000 000 d). You can do repeated washes and g-forces are considerably lower than with UC

Thank you so much for all these documented answers. Here are some comments to these answers:

@ Christoph Metzner: archaea and their viruses can grow in hostile environments (but not only) but are not as resistant as one may think. For instance, our virus is not stable in the growth medium for more than 30 minutes (ph2, 80°), which means that we must neutralize the medium prior to purify it. Our host is highly sensitive to detergent traces, all labware have to be washed manually.

Despite this, our actual purification procedure, is to neutralize the culture, centrifuge briefly to pellet the cells, filtrate the supernatant and finally ultracentrifuge to pellet the viruses. EM with these viruses is OK in negative staining, but fails the quality required for cryo-EM. Hence my question on purification procedures used for envelopped viruses.

From all yours answers, I would give a try on CsCl gradient, hoping that the virus will stand the centrifugational force. By the way, are the conditions similar to DNA CsCl purification (nVT or VTI rotors, 60K) or is it less stringent ?

Meanwhile, we can also test the Sartorius Stedim columns (which I suppose are the Vivaspin columns?) as well as CIM monolithic columns suggested by Andrej Steyer.

Do not filter the virus containing fluid neither pellet the virus, just use cell free (cells peletted by max 1200xg spin) virus containing medium to fix in gluteraldehyde and proceed for cryo-EM. This assures intact virus morphology though the virus concentration may be less and one has to look for a virus in several preps.

have a look at http://www.axis-shield-density-gradient-media.com/virusindexes.htm.

They have a wide variety of protocols

I don't prefer CsCl purifications, because for viable virus there is an extra step than for DNA preps. You have to dialyze the collected banded virus in a large volume of some neutral pH buffer so that the CsCl can diffuse out. Otherwise, the virus is not infectious. Moreover, the CsCl will kill whatever cells you're infecting it with. The buffer and the size of the dialysis membrane you choose will depend on the size of the virus.

I work on flaviviruses. The first important step is to have a viral culture of quality: do not let the virus overgrow, harvest when the cells are in good shape, do multiple harvests from the same culture is the virus is growing slowly or if starting with a low moi in order to avoid partial damaging of the 1st secreted particles. You can check the quantity of virus by qPCR or titration before pooling the fractions. If your virus is sensitive to pH, do not wait for your pH indicator to turn before harvesting. For flaviviruses (about 50 nM diameter) 1 mg of purified virus is needed for good images in cryo-microscopy, in order to be able to count about 100 particles in one field. In terms of PFU, it means 0.5 mL at a concentration of about 10e11 PFU/mL. So before performing the sucrose gradient, we concentrate the supernatant by PEG precipitation (7.5%) for at least 3 hours or overnight at 4°C, and high-speed centrifugation. The pellet in re-suspended in a small volume in an appropriate buffer ( PBS, CA+Mg+) and dialyzed (or ultra-filtrated if the volume needs to be reduced again) at 4°C against the same buffer. the virus is then loaded on an appropriate sucrose-gradient. The pH of the sucrose solutions is also checked and contains Ca+Mg+ ions to maintain the integrity of the lipid membrane. When analyzing the gradients fraction, a sharp peak is indicator of a homogeneous population. If not the case a second gradient purification might improve the curve. If possible, he samples should be analyzed immediately after pooling and dializing the fraction of interest. Storage before analysis is a very critical point and will depend on the characteristics of the virus. At 4°C, there is a risk of aggregation. At -70°C, cryoprotectors like sucrose cannot be used (interferes with the quality of the ice), and freezing must be done as fast as possible. It is better to freeze aliquots to avoid freeze-thawing.

Thanks a lot for all these new informations. Meanwhile, reading several papers and reading your answers, made me change my mind from CsCl to sucrose gradient. The major problem with CsCl is that if we want to try EM on the early steps of infection it may interfere. I will let you know how we progress in the purification.

CsCl gradients are not recommended for enveloped viruses, unless you want to purify the core of the virus: they dissociate the lipid bilayer

You might want to try potassium tartrate/glycerol gradients (see attachment) - this was used to purify dengue virus for structure determination by cryo-EM (kuhn et al, Cell, 2002).

Thank you Joseph for this suggestion

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

easy protocol + you do not loose surface glycoproteins due to shearing in the gradient... the only thing you may need to do is remove the gradient- medium/purify the sample, as iodixanol may interfere with your cryo-EM protocol.

I have a related question to Marie-Claude's; I am working with environmental samples (biogas reactors) with and heavy bacterial, fungal and protozoa.

I want to make a virus preparation for a metagenomic study. What is the maximum g I should use to sediment the cells (bacteria - protozoa) without also sedimenting any viruses the may be present.

The problem I have at the moment is the clogging of my 0.45um membranes.

This is mainly a matter of time. In an centrifugation with up to 10.000g you won´t pellet virus within 15min (this is what we use to pellet bacteria). Stuff clogging your membrane might be already bigger, this will be pelleted at 1000g within a few minutes.

Perhaps you try a step wise pelleting.

Guys,

I have adult insects naturally infected with a new Flavivirus. I would like to purify virus particles from these insects. Do you have any protocol that you can share it with me? Thank you