techniques of purification of viruses

1. Dr. R. S. Jadhav
Department of Microbiology
VNBN Mahavidyalaya, Shirala

2.  To study of virus structure, reproduction of virus,
vaccine production of virus etc., requires pure viruses.
 A number of techniques are available for purification of
viruses.
 Due to the virus futures such as,Virus size is larger than
proteins, more stable than cell components and
presence of surface proteins, techniques used for virus
purification are similar to the techniques used for
isolation and purification of proteins and cell
organelles.

3.  Density gradient centrifugation based on sedimentation of
particles in a density gradient medium.
 This method separates particles based on their buoyant
densities.
 This method give much better separation than differential
centrifugation.
 There are two methods, namely:
 1) Zonal centrifugation
 2) Isopycnic or equilibrium density gradient centrifugation.

4.  Sample is centrifuge in a performed gradient.
 A density gradient is prepared in a centrifuge tube before
centrifugation.
 Different substances are used as gradient material such as sucrose,
cesium chloride,cesium acetate, glycerol, ficoll and ludox(silica gel).
 Density gradient is prepared by layering solutions of continuously
decreasing densities over each other.
 The highest density solution is at the bottom of centrifuge tube
whereas lowest density solution is at top of centrifuge tube.
 The virus sample to be separated is layered on top of gradient.
 Centrifugation is carried out at a relatively low speed for a short time
(1 to 3 hrs).
 The sample particles travel the steep gradient based on their
sedimentation rate and form discrete zones.
 This method separates particles, which differ in size but not in
density.

5.  The largest viruses move faster down the gradient.
 The centrifuge run is terminated before any of the
zones reaches the bottom of gradient.
 The zone in the gradient are stable due to absence of
diffusion and convection currents. Collecting fractions
from bottom of the tube isolates various zones.

6.  In this method the density gradient forms during
centrifugation.
 The sample to be separated is dissolved in a solution of
cesium chloride or cesium sulfate.
 This mixture is distributed uniformly in a centrifuge
tube.
 Centrifuge is carried out at a high speed for long
time(48 hrs).
 The cesium salt redistributed under the influence RCF
to form a continuously increasing gradient from top to
bottom.

7.  The sample particles move the gradient at a
region, where the gradient density is equal to
their respective densities and form discrete
zones.
 This method separates particles, which differ in
density but not in size.
 Viruses can be separated from other particles
only slightly different in density.

8.  Like proteins, viruses can be purified through
precipitation with conc. Ammonium sulphate or
ethanol or polyethylene glycol.
 Initially sufficient conc. Of ammonium sulphate is
added so that virus will precipitate.
 In this manner more and more purified
precipitation are obtained.
 The precipitated viruses are collected by
centrifugation.

9. Thanking you