1. Virus purification and analysis using OptiPrep™ - competitive media
Sucrose
Glycerol
Caesium chloride
Traditional virus isolation media

2. SOLUTION HANDLING
Density solutions prepared by diluting OptiPrep™ with culture medium or any balanced salt solution (Application Sheet V01)
Sterile solution
CsCl/sucrose solutions require lengthy preparation times and sterilization
CsCl is expensive
Considerable time-saving if OptiPrep is used

3. CsCl PROBLEMS
Big losses in viral infectivity; low recoveries; high particle:infectivity ratios
Solutions have very high osmolality: viruses lose water; high virus density; high [CsCl] required for gradients
Highly toxic to cells: must be dialyzed prior to re-infection of cells
Solutions ionic and corrosive: samples must be dialyzed before electrophoresis or HPLC
CsCl is basically a highly toxic heavy metal salt

4. SUCROSE PROBLEMS (I)
Solutions are hyperosmotic: virus density in sucrose usually higher than in iodixanol
Solutions are viscous: slow sedimentation of virus particles
Highly toxic to cells: must be dialyzed prior to re-infection of cells
Dialysis adds an overnight step.

5. OPTIPREP – NO PROBLEMS Solutions are isoosmotic: virus density is low
Solutions have low viscosity: rapid sedimentation of virus particles
Is non-toxic to cells
Has little or no effect on virus infectivity
Cells can be re-infected and electrophoresis and HPLC performed without dialysis
Only for electron microscopy is it necessary to remove the iodixanol; either dialysis or ultrafiltration are recommended – for more information see Application Sheet V27.

6. Virus banding density Virus CsCl Sucrose Iodixanol Epstein-Barr 1.26
1.14
Semliki Forest
1.18
Poliovirus
1.34
1.22
Measles
1.21
1.16
Virus banding density always lower in iodixanol than in CsCl; in sucrose differences are smaller

7. % iodixanol, sucrose, glycerol
Effect of medium on PM2 infectivity
Kivela et al (1999)Virol. 262, 10 20 30 40 50 60
% iodixanol, sucrose, glycerol
1011
1012
1.1
1.3
1.5
1.7
CsCl g/ml
Iodixanol
Glycerol
CsCl
Sucrose
Infectivity
PM2 is a bacteriophage.

8. SUCROSE PROBLEMS (II): for enveloped viruses, particularly retroviruses
Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradients
Infectivity recoveries are x greater in iodixanol than in sucrose
Hydrodynamic shearing forces at the surface of the virus particles cause the loss of glycoproteins when the they move through a viscous sucrose gradient

9. rAAV in discontinuous gradient (V04)
Zolotukhin S. et al (1999) Gene Therapy, 6,
15% iodixanol
in 1 M NaCl
25% iodixanol
40% iodixanol
54% iodixanol
Virus fluid
350,000g 1 h
proteins
adenovirus
rAAV
The most widely used method for purification of one of the most widely used viruses in gene therapy studies. The NaCl in the top layer prevents adsorption of soluble proteins to the rAAV and aggregation at the top two interfaces.

10. rAAV in continuous gradient (V04)
Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10,
Opti-Prep
Virus
fluid B
Gradient Master
An alternative method for the purification of rAAV is a pre-formed continuous gradient. This illustrates one of the advantages of using the Gradient Master, or other similar device for the creation of a continuous gradient when handling any type of virus. The virus fluid is simply layered over the same volume of OptiPrep (or 50% iodixanol); the tube sealed and the gradient created by controlled mixing in 2-3 min. It is thus a very safe method particularly for potentially infective viruses.

11. Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time
Density (g/ml)
Fraction Number 1 3 5 7 9
1.07
1.09
1.11
1.13
2 min
2.5 min
3.0 min
Some examples of the profiles of gradients formed in the Gradient Master.

12. 3 Formats for separation of particles according to their density1 2 3
In this method the particles in the virus suspension are distributed throughout the gradient and potential interactions of virus and non-virus particles at interfaces are avoided (Format 3) and the Gradient Master is the easiest means of creating this format.
For more information on the theory of density gradient centrifugation see Training File 1 (Slides 16-24).

13. rAAV in continuous gradient (V04) Hermans, W. T. J. M. C
rAAV in continuous gradient (V04) Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10,
OptiPrep
Virus
fluid
rAAV
membranes
348000g
3 h
In this continuous iodixanol gradient the rAAV bands about ¾ the way down the tube and is well resolved from other particulate contaminants.

14. Comparison of rAAV in CsCl and OptiPrep™
Pre-gradient
Gradient
% Recov’ry
VP/ICU
(NH4)2 SO4
CsCl
0.1-60
OptiPrep
26-120
Cellufine-SO4
25-91
The recoveries of rAAV in iodixanol are higher and more reliable than in CsCl gradients and the particle:infectivity ratios are up to 100,000 times lower.
Data from Hermans, WTJMC et al (1999) Human Gene
Therapy, 10,

15. Purification of HIV-1 (V06)
Dettenhoffer, M & Yu, X-F (1999) J. Virol.,
RT (cpmx10-5/ml)
250,000g for 1.5 h
6-18% iodixanol
Microvesicles
Vif gene
Soluble proteins
As a contrast to the rAAV methods which employ either a discontinuous or continuous gradient to purify the vector according to its density, here is another widely applied option – a shallow sedimentation velocity iodixanol gradient for studying the processing of viruses – originally applied to HIV-1 put now applied to a wide range of enveloped viruses. The virus is identified in the gradient by reverse transcriptase activity (RT). The virus is well separated from other particulate and soluble components. Being a sedimentation velocity gradient, its efficacy is dependent on the use of relatively small volumes of virus containing material. Larger scale preparations in which the virus is banded according to its buoyant density are also given in Application Sheet V06.
For more information on the harvesting of banded material in density gradients see Training File 2 (Slides 19-24).

16. Use of self-generated iodixanol gradients in vertical rotors for virus purification and analysis
Like CsCl, iodixanol can form self-generated gradients at high g-forces and such gradients are also a widely used and a very simple option for virus purification and analysis. For more information about the formation of self-generated gradients see Training File 2 (Slides 10-18).

17. Virus purification flow-chart – without virus pelleting1 2 3
50,000g 1h 5 4
350,000g
1-3h
A strategy which avoids virus pelleting (pelleting reduces the infectivity of many viruses) is shown in the slide. 1-2: The virus is banded on to 2-3 ml of a dense iodixanol cushion; 2-3: All of the supernatant is removed except for 2-3 ml. 3-4: The remaining solutions are mixed and transferred to a tube for a vertical rotor. 4-5: The virus is banded in a self-generated gradient.

18. Purification of Herpes virus in self-generated 25% iodixanol gradient (V05)
Fraction Number
Log infectivity
Density (g/ml) 1 3 5 7 9 11 13 15 2 4 6
1.05
1.1
1.15
1.2
1.25
1.3
Infectivity
Density
An example of Herpes virus banding in a self-generated gradient.

19. Purification of retrovirus: self-generated gradient (V09)
Moller-Larsen A & Christensen, T. (1998) J. Virol Meth., 73,
Fraction number
OD(PERT) x 10-2
Density (g/ml) 1 3 5 7 9 11 13 15 17 19 10 20 25 30
1.05
1.1
1.15
1.2
1.25
MS1533
MS1874
Density
An example of endogenous retrovirus virus banding in a self-generated gradient.

20. OptiPrep™Application Sheets for:
Adenovirus
Ebola virus
Foamy virus
Herpes simplex virus
HIV-1
Human papillomavirus
Lassa virus
Murine leukemia virus
Murine oncornavirus
Norwalk virus
Polyoma virus
rAAV
Rabies virus
Rous sarcoma virus
Retroviruses
Semliki Forest virus
SARS Corona virus
Vaccinia virus
Some of the viruses that have been purified using OptiPrep

21. Publications database on viruses
OptiPrep (since 1994) over 600
Nycodenz® (since 1984) approx 70
Using either the Applications CD or the website:
www. axis-shield-density-gradient-media.com
Follow the instructions to access the relevant Index
Click on the virus of interest
Abstracts of all the papers reporting the use of OptiPrep or Nycodenz for virus isolation and analysis can also be accessed from the same CD or website.