Types of sedimentation medium:

 

A. Sucrose cushions or gradient - A fixed concentration or a linear gradient of sucrose is used. Increasing the density and viscosity of the medium decreases the rate at which virus sediments through them. In g eneral a "cushion" of sucrose is prepared at the bottom of the centrifuge tube and the sample containing virus is overlaid over the cushion. Since most viruses have greater densities than sucrose, separation is based on S values. This method can be used to separate molecules with relatively close S values. Sometime glycerol is also used in place of sucrose.

 

B. CsCl 2 gradient centrifugation - A linear gradient of CsCl 2 in buffer is prepared in the ultracentrifuge tube. As the concentration of the CsCl 2 is increased the density of the medium increases in the tube so that density is low at the top and high at the bottom. Viral particle centrifuged through this medium will form a band at a position equal to their buoyant density. These are useful to separate viruses of different densities. Limitation of this method is that CsCl 2 can permanently inactivate some viruses.

 

6.2.2. Other techniques for separation:

Viruses can also be separated by electrophoresis and column chromatography but these are not the preferred way to separate virus while sometimes they are used to separate viral nucleic acids or proteins. Both the methods separate the virus on the basis of charge and/or size. Virus contains a variety of charged macromolecule on its surface which contributes to its electrophoretic mobility or ion-exchange characteristics. Viruses are sometimes ligated with the charged group to be separated by ion exchange chromatography. Molecular sieve chromatography can also be used to purify the viruses where large pores are formed with the help of special agarose through which virus particles can enter.

 

6.3. Purity of viruses:

Many methods are used to assess the purity of virus. The ratio of UV absorption at 260 and 280 nm during a spectrophotometric analysis (260/280) is a characteristic feature to measure the purity of a virus sample and is dependent on the amount of nucleic acid and protein present in the virion. Serological methods such as enzyme-linked immunosorbent assay (ELISA), radioimmuno precipitation assay (RIPA), western blot, virus neutralization test (VNT), and complement fixation are also used to check the puirity of a virus sample. These methods require antibodies specific to viral proteins that may be monoclonal (single type of antibody specific to a single viral protein) or polyclonal (several different antibodies that may recognize several viral proteins or epitopes). Plaque assay is also performed in order to isolate the single colony from a pool of quasispecies viruses.