Purification of viruses

techniques of purification of viruses

1. Purification Viruses 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. Introduction

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. 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. 1) Zonal centrifugation

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. 2) Isopycnic or equilibrium density gradient centrifugation

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. 2. Precipitation

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