Isolation and purification of viruses

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 I nvolves 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 MgCl 2 The virus particles come out because they have greater affinity to the medium