Purification and Identification of Viruses
المؤلف:
Stefan Riedel, Jeffery A. Hobden, Steve Miller, Stephen A. Morse, Timothy A. Mietzner, Barbara Detrick, Thomas G. Mitchell, Judy A. Sakanari, Peter Hotez, Rojelio Mejia
المصدر:
Jawetz, Melnick, & Adelberg’s Medical Microbiology
الجزء والصفحة:
28e , p424-425
2025-10-21
81
Purification of Virus Particles
Pure virus must be available in order for certain types of studies on the properties and molecular biology of the agent to be carried out. For purification studies, the starting material is usually large volumes of tissue culture medium, body fluids, or infected cells. The first step frequently involves concentration of the virus particles by precipitation with ammonium sulfate, ethanol, or polyethylene glycol or by ultrafiltration. Hemagglutination and elution can be used to concentrate orthomyxoviruses. After concentration, virus can be separated from host materials by differential centrifugation, density gradient centrifugation, column chromatography, and electrophoresis.
More than one step is usually necessary to achieve adequate purification. A preliminary purification will remove most nonviral material. This first step may include centrifugation; the final purification step almost always involves density gradient centrifugation. In rate-zonal centrifugation, a sample of concentrated virus is layered onto a preformed linear density gradient of sucrose or glycerol, and during centrifugation the virus sediments as a band at a rate determined primarily by the density of the virus particle.
Viruses can also be purified by high-speed centrifugation in density gradients of cesium chloride, potassium tartrate, potassium citrate, or sucrose. The gradient material of choice is the one that is least toxic to the virus. Virus particles migrate to an equilibrium position where the density of the solution is equal to their buoyant density and form a visible band.
Additional methods for purification are based on the chemical properties of the viral surface. In column chromatography, virus is bound to a substance such as diethylaminoethyl or phosphocellulose and then eluted by changes in pH or salt concentration. Zone electrophoresis permits separation of virus particles from contaminants on the basis of charge. Specific antisera also can be used to remove virus particles from host materials.
Icosahedral viruses are easier to purify than enveloped viruses. Because the latter usually contain variable amounts of envelope per particle, the viral population is heterogeneous in both size and density.
It is very difficult to achieve complete purity of viruses. Small amounts of cellular material tend to adsorb to particles and copurify. The minimal criteria for purity are a homogeneous appearance in electron micrographs and the failure of additional purification procedures to remove “contaminants” without reducing infectivity.
Identification of a Particle as a Virus
When a characteristic physical particle has been obtained, it should fulfill the following criteria before it is identified as a virus particle:
1. The particle can be obtained only from infected cells or tissues.
2. Particles obtained from various sources are identical regardless of the cellular origin in which the virus is grown.
3. Particles contain nucleic acid (DNA or RNA), the sequence of which is not the same as the species of host cells from which the particles were obtained.
4. The degree of infective activity of the preparation varies directly with the number of particles present.
5. Destruction of the physical particle by chemical or physical means is associated with a loss of viral activity.
6. Certain properties of the particles and infectivity must be shown to be identical (eg, their sedimentation behavior in the ultracentrifuge and their pH stability curves).
7. Antisera prepared against the infectious virus should react with the characteristic particle and vice versa. Direct observation of an unknown virus can be accomplished by electron microscopic examination of aggregate formation in a mixture of antisera and crude viral suspension.
8. The particles should be able to induce the characteristic disease in vivo (if such experiments are feasible).
9. Passage of the particles in tissue culture should result in the production of progeny with biologic and antigenic properties of the virus.
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