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Section 5.1
Fundamentals of
Purification of Plant Viruses
Knowledge about a virus, before being purified, is very limited. Very pure
preparations of viruses are required in order to carry out chemical,
physical, and other biological studies. There are numerous purification
procedures that can be adapted to many of the viruses that infect plants.
However, there are several different purification systems that can be
selected for use according to the type of virus.
Virus particles of this group contain one molecule of central nucleic acid
(which contains the genetic code) surrounded by a protein capsid. Rod
shaped viruses such as tobacco mosaic and some spherical viruses such
as potato leafroll and sowbane mosaic belong to this group.
Group 2. Multicompound Nucleoproteins
Besides the protein capsid that encloses the nucleic acid, these viruses
have an external membrane composed of lipids. These lipid membranes
are very delicate and are also easily damaged, which can cause the
inactivation of the particles. The procedures of virus purification in Group
1, which are aimed at destroying membranes, are not suitable for these
viruses. More gentle methods must be used.
Group 5. Viroids
Viroids, like the potato spindle tuber viroid (PSTVd), do not have a protein
capsid. They are free molecules of nucleic acid. The methods of
purification are based on methods used for the isolation of nucleic acids.
They cannot be isolated using any of the procedures used for the four
groups mentioned above.
The facility and speed of spreading in a host plant and the growth stage
in which they can be inoculated are very important factors to take into
account during selection.
The selected host plants selected should not contain great quantities of
tannins, gums, or phenolic compounds, because such compounds
interfere with virus purification. Generally, great quantities of these
compounds are found in ligneous plants, which makes them
inappropriate for purification.
Certain plant viruses multiply in the leaves during a long period of time,
while others reach a concentration peak within a short period. With
certain viruses, concentration increases up to a certain level and then
starts to decrease. It is important to harvest the leaves when the
concentration level is close to the maximum. It is also important to
consider that virus concentration in different parts of the plant can vary
considerably.
Extraction Medium
1. pH
7. Detergents
Triton x-100 can be used to help to liberate the viruses from those
insoluble cellular components such as membranes.
8. Enzymes
1. Freezing
2. Heating
3. Organic Solvents
Extract clarification
The first things to eliminate are any particles larger than the virus, such
as chloroplasts, mitochondrias, etc. This is achieved by filtering the
extract.
An extract can be filtered through some layers of filter paper, many layers
of gauze, a fine nylon net, or glass wool to eliminate pieces of tissue and
unfragmented cells. The filtration can also be carried out through carbon,
diatomacrous earth, bentonite, cellulose powder, or Celite.
The use of glass wool and gauze is only effective to filter membranes and
big pieces of tissue. The filtrate still contains chloroplasts. A 10,0000-g
centrifugation for 10 minutes is usually enough to remove them. This
treatment produces sediment, which contains most of the big debris from
the plant. The viruses, certain kinds of ribosomes, particles smaller than
the viruses, proteins, and dissolved salts remain in solution.
The procedure used is chosen according to the stability of the virus and
the contaminants present in the clarified extract.
2. Crystallization
3. Isoelectric precipitation
4. Electrophoresis
7. Ultracentrifugation
9. Serological methods
11. Microfilters
This technique has been widely used for the purification of animal
viruses. Systems of filters with pores of different sizes can be
arranged so that pre-filtration (to remove the biggest particles that
can clog filters with small pores) and virus concentration occur
simultaneously. This method is suitable for globular molecules and
for inflexible rod-shaped viruses such as the tobacco mosaic. It is
not recommended to use ribbon-shaped flexible molecules,
because they can be broken by the torsion force of filtration and
their fragments can clog the filters. Another disadvantage is that
viruses can stick on the filters of pre-filtration. One way of avoiding
virus loss consists in using filters with a pore diameter at least ten
times bigger than the size of the virus to be filtered.