Virus Purification and Assays
Virologists must be able to purify viruses and accurately determine their concentrations in order to study virus structure, reproduction, and other aspects of their biology. These methods are so important that the growth of virology as a modern discipline depended on their development.
Purification makes use of several virus properties. Virions are very large relative to proteins, are often more stable than normal cell components, and have surface proteins. Because of these characteristics, many techniques useful for the isolation of proteins and organelles can be employed in virus isolation.
Four of the most widely used approaches are: –
- Differential and density gradient centrifugation.
- Precipitation of viruses.
- Denaturation of contaminants.
- Enzymatic digestion of cell constituents.
Differential and density gradient centrifugation
- Host cells in later stages of infection that contain mature virions are used as the source of material.
- Infected cells are first disrupted in a buffer to produce an aqueous suspension or homogenate consisting of cell components and viruses.
- Viruses can then be isolated by differential centrifugation, the centrifugation of a suspension at various speeds to separate particles of different sizes.
- Usually the homogenate is first centrifuged at high speed to sediment viruses and other large cellular particles, and the supernatant, which contains the homogenate’s soluble molecules, is discarded.
- The pellet is next resuspended and centrifuged at a low speed to remove substances heavier than viruses.
- Higher speed centrifugation then sediments the viruses. This process may be repeated to purify the
virus particles further.
- Viruses also can be purified based on their size and density by use of gradient centrifugation.
- A sucrose solution is poured into a centrifuge tube so that its concentration smoothly and linearly increases between the top and the bottom of the tube.
- The virus preparation, often after purification by differential centrifugation, is layered on top of the gradient and centrifuged.
- The particles settle under centrifugal force until they come to rest at the level where the gradient’s density equals theirs (isopycnic gradient centrifugation).
- Viruses can be separated from other particles only slightly different in density.
- Gradients also can separate viruses based on differences in their sedimentation rate (rate zonal gradient centrifugation).
- When this is done, particles are separated on the basis of both size and density; usually the largest virus will move most rapidly down the gradient.
- Viruses differ from one another and cell components with respect to either density (grams per milliliter) or sedimentation coefficient(s).
- Thus these two types of gradient centrifugation are very effective in virus purification.
Precipitation of viruses
- Viruses, like many proteins, can be purified through precipitation with concentrated ammonium sulfate.
- Initially, sufficient ammonium sulfate is added to raise its concentration to a level just below that which will precipitate the virus.
- After any precipitated contaminants are removed, more ammonium sulfate is added and the precipitated viruses are collected by centrifugation.
- Viruses sensitive to ammonium sulfate often are purified by precipitation with polyethylene glycol.
Denaturation of contaminants
- Viruses frequently are less easily denatured than many normal cell constituents.
- Contaminants may be denatured and precipitated with heat or a change in pH to purify viruses.
- Because some viruses also tolerate treatment with organic solvents like butanol and chloroform, solvent treatment can be used to both denature protein contaminants and extract any lipids in the preparation.
- The solvent is thoroughly mixed with the virus preparation, then allowed to stand and separate
into organic and aqueous layers.
- The unaltered virus remains suspended in the aqueous phase while lipids dissolve in the organic phase.
- Substances denatured by organic solvents collect at the interface between the aqueous and organic
Enzymatic digestion of cell constituents
- Cellular proteins and nucleic acids can be removed from many virus preparations through enzymatic degradation because viruses usually are more resistant to attack by nucleases and proteases than are free nucleic acids and proteins.
- For example, ribonuclease and trypsin often degrade cellular ribonucleic acids and proteins while leaving virions unaltered.
The quantity of viruses in a sample can be determined either by counting particle numbers or by measurement of the infectious unit concentration. Although most normal virions are probably potentially infective, many will not infect host cells because they do not contact the proper surface site. Thus the total particle count may be from 2 to 1 million times the infectious unit number depending on the nature of the virion and the experimental conditions. Virus particles can be counted directly with the electron microscope.
In one procedure the virus sample is mixed with a known concentration of small latex beads and sprayed on a coated specimen grid. The beads and virions are counted; the virus concentration is calculated from these counts and from the bead concentration. This technique often works well with concentrated preparations of viruses of known morphology. Viruses can be concentrated by centrifugation before counting if the preparation is too dilute. However, if the beads and viruses are not evenly distributed (as sometimes happens), the final count will be inaccurate.
The most popular indirect method of counting virus particles is the hemagglutination assay. Many viruses can bind to the surface of red blood cells. If the ratio of viruses to cells is large enough, virus particles will join the red blood cells together, forming a network that settles out of suspension or agglutinates. In practice, red blood cells are mixed with a series of virus preparation dilutions and each mixture is examined. The hemagglutination titer is the highest dilution of virus (or the reciprocal of the dilution) that still causes hemagglutination. This assay is an accurate, rapid method for determining the relative quantity of viruses such as the influenza virus. If the actual number of viruses needed to cause hemagglutination is determined by another technique, the assay can be used to ascertain the number of virus particles present in a sample.
A variety of assays analyze virus numbers in terms of infectivity, and many of these are based on the same techniques used for virus cultivation. For example, in the plaque assay several dilutions of bacterial or animal viruses are plated out with appropriate host cells. When the number of viruses plated out are much
fewer than the number of host cells available for infection and when the viruses are distributed evenly, each plaque in a layer of bacterial or animal cells is assumed to have arisen from the reproduction of a single virus particle. Therefore a count of the plaques produced at a particular dilution will give the number of infectious virions or plaque-forming units (PFU), and the concentration of infectious units in the original sample can be easily calculated. Suppose that 0.10 ml of a 10–6 dilution of the virus preparation yields 75 plaques. The original concentration of plaque-forming units is: –
PFU/ml (75 PFU/0.10 ml)(106) = 7.5 × 108
Viruses producing different plaque morphology types on the same plate may be counted separately. Although the number of PFU does not equal the number of virus particles, their ratios are proportional: a preparation with twice as many viruses will have twice the plaque-forming units.The same approach employed in the plaque assay may be used with embryos and plants. Chicken embryos can be inoculated with a diluted preparation or plant leaves rubbed with a mixture of diluted virus and abrasive. The number of pocks on embryonic membranes or necrotic lesions on leaves is multiplied by the dilution factor and divided by the inoculum volume to obtain the concentration of infectious units.
When biological effects are not readily quantified in these ways, the amount of virus required to cause disease or death can be determined by the endpoint method. Organisms or cell cultures are inoculated with serial dilutions of a virus suspension.The results are used to find the endpoint dilution at which 50% of the host cells or organisms are destroyed. The lethal dose (LD50) is the dilution that contains a dose large enough to destroy 50% of the host cells or organisms. In a similar sense, the infectious dose (ID50) is the dose which, when given to a number of test systems or hosts, causes an infection of 50% of the systems or hosts under the conditions employed.