Virus isolation in embryonated eggs, cell cultures and animals Purification by centrifugation, chromatography and electrophoresis 3d models such as organoid cultures is not discussed

1. ISOLATION AND PURIFICATION
OF VIRUSES
Darshan

2. Isolation of Viruses
Sample collection
 Symptoms as criteria
 Animal viruses: Nasal swabs, stool samples,...
 Usage of transport medium: eg Phosphate broth with 0.5% gelatin
 Plant and insect viruses: crushed parts to obtain samples
 Concentration of viruses

3. Embryonated
eggs
• Sterile and has wide variety of tissue
and cavity fluid
• Eggs are candled, disinfected and
marked
• A hole is drilled and virus is
injected via appropriate route, and
covered with wax
• Virus growth may result in the
death of the embryo (e.g.
encephalitis virus), the production
of plaques on the chorioallantoic
membrane (e.g. herpes, smallpox,
vaccinia), the development of
hemagglutinins in embryonic fluids
or tissues (e.g. influenza)

4. Laboratory Animals
 Live animals such as monkeys, mice, rabbits, guinea pigs, ferrets
are widely used for cultivating virus
 Monkeys were used for the isolation of Poliovirus
 Mice are the most widely employed animals in virology
 Different routes of inoculation in mice- intracerebral,
subcutaneous, intraperitoneal or intranasal
 After inoculation animals are observed for signs of disease,
visible lesions or is killed so that infected tissues can be examined
for virus

5. Cell cultures
Primary cell lines
 Limited life span – 5 to 20 cell divisions
 Derived from monkey kidneys, human foreskins, respiratory epithelium..
Continuous cell line
 Consists of a single cell type that can be propagated indefinitely in culture
 Immortal lines derived from tumor tissues eg: HeLa cells
Diploid cell strains
 Consists of a homogenous population of a single type
 100 time divisions before dying eg: human diploid fibroblast cells

6. Purification of Viruses
 Separation of virus from host tissues and cell organelles
Centrifugation
 Involves the application of centrifugal force to separate particles from a solution
according to their size, shape, density, viscosity of the medium and rotor speed

7. Differential Centrifugation
 Involves alternating cycles of low-speed centrifugation, after which most of the virus is still in the
supernatant, and high-speed centrifugation, after which the virus is in the pellet
A crude preparation of virus containing host
debris is subjected to low-speed/short-time
centrifugation (e.g. 10 000 g/20 minutes) followed
by high-speed/long time centrifugation (e.g.
100000 g/2 hours). This cycle can be repeated to
obtain a higher degree of purity. The final pellet
containing partly purified virus is resuspended in
a small volume of fluid.

8. Density gradient centrifugation
Rate zonal centrifugation
 Particles move through the gradient at a rate
determined by its sedimentation coefficient
 Preformed gradient with increasing density –
sucrose
Isopycnic centrifugation
 Gradient is formed after centrifugation – CsCl
 Particles move to point where their bouyant
density equals that part of gradient and form
bands

9. Electrophoresis
 Based on charge and/or size
 Charge on virus particles contribute to electrophoretic mobility
 The electrophoresis in a density gradient column (a liquid medium, usually
sucrose) is more commonly used because a much larger volume can be
processed
 Gel electrophoresis- small quantity of sample to be used
 Zonal electrophoresis- large quantity of sample can be used in a density
gradient column (a liquid medium, usually sucrose)

10. Chromatography
 Used for final purification of partially purified samples and proteins
 Dependent on surface properties of viruses
 Solid substances such as calcium and aluminum phosphate packed on columns
of glass tubes onto which impure virus suspension is poured
 Unadsorbed particles are removed by washing
 Adsorbed viruses are removed washing with large volumes of specific medium
such as MgCl2
 The virus particles come out because they have greater affinity to the medium