Viruses can only replicate inside living cells and infect all forms of life. They are submicroscopic and were first observed in 1892 as infecting tobacco plants. To extract viruses, infected plant leaves are homogenized and filtered to obtain a crude sap containing viruses. This sap is then centrifuged at increasing speeds to separate and pelletize the viruses. The purified virus pellet can then be re-suspended in buffer. Further purification techniques include precipitation using alcohol or ammonium sulfate to remove plant proteins, or gel filtration to separate viruses from other particles.

1. BY
R.LOKESH
I -M.sc.,Agriculture
Plant pathology
Annamalai university

2. VIRUS
• A virus is a submicroscopic infectious agent that replicates only inside the living cells of
an organism.
• Viruses infect all life forms, from animals and plants to microorganisms,
including bacteria and archaea.
• Since Dmitri Ivanovsky's 1892 article describing a non-bacterial pathogen infecting tobacco plants
and the discovery of the tobacco mosaic virus by Martinus Beijerinck in 1898

4. Growing and extraction of virus
• Infected leaves are thoroughly homogenized in water or
preferably in phosphate, borate or citrate buffer in an
electric grinder or in a mortar with pestle
• Tissue homogenate is strained through a piece of muslin
cloth (or cheese cloth).

5. FRACTIONATING VIRUS CONTAINING EXTRACT
• Crude sap which comes out and contains virus is collected and then
poured into centrifuge tube.
• The tube is spun at low-speed (3000-17000 g).
• As a result, the crude sap differentiates into supernatant and a pellet.
• The pellet is discarded and the supernatant with virus is collected

6. • The supernatant with virus is poured into centrifuge tube.
• The tube is placed in fixed-angle-rotor of ultracentrifuge and spun
at high speed (40000- 150000 g).
• After the tube settles, the virus sediments and forms tiny pellet at
the bottom of the tube and a supernatant over it.
• Supernatant is discarded and the pellet of virus is mixed with a
buffer and stirred with rod so that it re-suspends in buffer.

12. • The two precipitating agents most commonly used are alcohol
and ammonium sulphate.
• The alcohol can be used for precipitating either the virus or,
the extraneous plant proteins according to the particular virus
being used or the strength of the alcohol.
• The amount of amonium sulphate used is usually one third
saturation.
PRECIPITATION OF THE VIRUS.

14. • By this method the virus suspension is allowed to percolate
slowly down a long tube filled with agar or 'Sephadex’.
• Very small particles enter the pores of the agar granules, but
larger ones such as virus particles are unable to do this and will
flow down through the column around and between the gel
granules with the movement of liquid through the column.
• The virus thus arrives at the base of the column and can be col-
lected before the arrival of the pigment and other small
particles.
GEL FILTRATION.