CHAPTER SEVENTEEN: CULTIVATION AND PURIFICATION OF VIRUSES
Musa Alhaji Isah
17.1 Cultivation
Many viruses can be grown in cell cultures or in fertile eggs (embryonated eggs) under strictly controlled conditions or inoculation into suitable host animal.
17.1.1 Embryonated Eggs
These are more practical for cultivation of viruses, ethical and economic and for ease of handing and relative freedom from contaminants. A developing chick – embryos are immune – deficient thus favours the growth of viruses. Most viruses will grow or can be adapted to grow hi fertile eggs, and some may kill the embryo or may produce visible evidence of specific infection on the Choricallantoic Membrane (C.A.M). haemagglutinnating viruses in allantioc and amnionic fluids will cause haemagglutinnation when incubated with appropriate species of erythrocytes (red blood cells). According to Willey , J. M. et al., (2008), there are four routes of inoculating – eggs for viral cultivation:
- C.A.M. used for many pox and some herpes viruses.
- All nontoxic cavity used for ortho– paramyxo and rhabdo–viruses.
- All nontoxic cavity used for ortho–and paramyxo–viruses.
- Yolk sac used for many touaviruses.
17.1.2 Animal Host
Ideally, the natural host of a viruses or closely related species should be used for animal inoculation. This mode of viral cultivation is not always practical on ethical or economic ground, while there is also the possibility of latent infection with the virus under consideration. The route of inoculation of the animals is an important factor due to the specific affinity some viruses have for certain tissues, e.g. intracerebral–inoculation of mice with rabiesvirus: subcutaneous inoculation of swine vesicular disease virus into pigs. Animal may show clinical signs of infection and these must be observed, or biopsy material taken for examination. Necrosis must be conducted thoroughly and any microscopic abnormalities and histological changes noted. Serology may be necessary fro the presence of specially acquired antibodies.
Isolation or demonstration of the virus may be attempted by egg inoculation or tissue culture, and by electron microscopy. Neutralization of virus with a specific antiserum herb re inoculation of animals will, of course prevent the occurrences of infection.
The growth of virus in animal hosts is still used for primary isolation of certain viruses and for studies of the pathogenesis of viral disease and viral on cogenesis.
17.1.3 Cell Cultures
The availability of cell grown in – vitro has facilitated the identification and cultivation of newly isolated viruses and the characterization of the previously known ones. Cell cultures have been on the success since the advent of antibiotics and fungicides which have made it possible to prevent contamination of cultures. The introduction of trysin facilitated monolayer growth of cells. Chemically defined growth media have been produced to satisfy the nutritional requirements of many different types of cells . According to Medigan, M.T. et al., (2009). There are 3 basic types of cell culture:
(a) Primary cultures are made dispersing cells (usually with trysin) from freshly removed host tissues. In general, they are unable to grow for more than a few passages in culture e.g. Monkey Kidney cells and Human – amnion cells.
(b) Secondary cultures (semi – continuous cells) are also known as Diploid cells. They all undergone a change that allow their limited cultures (up to 50 passages) but which retain their normal chromosome pattern e.g Human embryo lung.
(c) Continuous cell lines are cultures capable of more prolonged, perhaps indefinite growth that have been derived from diploid cell lines or form malignant tissues. They invariably have altered and irregular numbers of chromosomes. The types of cell culture used for virus cultivation depend on the sensitivity of the e cells to a particular virus Continuous cell Lines is also referred to as Heteroploids cells lines e.g Hela cells derived from human cervical cancer.
Conclusion
Viruses can be cultivated via several means including embryonated eggs, animal host and cell cultures.
Summary
The best method for culturing viruses is cell cultures as even the most difficult virus can grow in cell lines suitable as host .
17.2 Purification
For purification, the starting material is usually large volumes of tissue culture medium, body fluids or infected cells, pure virus is important so as to have meaningful studies o the properties and molecular biology of the virion. The first frequently involved concentration of the virus particles by precipitation with ammonium and elution can be used to concentrate orthomyxoviruses. Once concentrated, virus can then be separated I mm materials by differential centrifugation, density gradient centrifugation, column chromatography and electrophoresis.
According to Pelczar, M.J. et al., (2001), more than one step is usually necessary to achieve adequate concentration. A preliminary purification will remove no n–virus material: the first step may include centrifugation while the final purification step almost always involves density gradient centrifugation. The band of purified virus may be detected by optical methods, by following radioactivity if the virus is radiolabelled, or by assaying infectivity.
Viruses can also be purified by high-speed centrifugation in density gradients of Cesium Chloride (CsC1), potassium tartarate, potassium citrate of Sucrose. The gradient material of choice is the one that is least toxic to the virus. The virus particles migrate to me equilibrium position where the density of the solution is equal to their buoyant density and form a visible band. Virus bands are harvested by puncture through the bottom of the plastic centrifuge lube and assayed for infectivity.
In column chromatography, virus is bound to substance such as DEAE or phosphocellulose and then eluted by changes in pH or Salt concentration. Zone electrophoresis permits the separation of virus particles from contaminant on the basis of charge. Specific antisera also can he used to remove virus particles from host materials
Icosahedral viruses are easier to purify than enveloped viruses because enveloped viruses contain variable amounts of envelope per particle, the virus population is heterogeneous in both size amid density. It is very difficult to achieve complete purity of viruses. Small amounts oc cellular materials tend to adsorb to particles and this co purity with the virion. The minimal criteria for purity are a homogenous appearance in electron micrographs and the failure of additional purification procedure to remove contaminants without reducing infectivity.
17.2.1 Centrifugation
Centrifugation as a purification and characterization procedure:
Ultracentrifuge: A centrifuge is capable of generating large centrifugal fields by rotating samples at 20,000 – 100,000 rpm. Centrifugal forces of greater than 100,000 X gravity can be generated.
17.2.2 Sedimentation Coefficient
§ Rate at which a macro molecule sediment under a defined gravitational force.
§ This parameter is influenced by both the molecular weight and shape of a macromolecule (larger and more spherical sed. Faster).
§ The basic unit is the Svedberg (S) which is 10-13 sec.
§ This value can be used to estimate molecular weights in conjunction with other values.
Buoyant density – Density at which a virus or other macromolecule neither sinks nor floats when suspended in a density gradient (e.g. CsC12 or source).
17.2.1.1 Types of sedimentation Medium
1. Aqueous Buffer (Water based) – Can be used to separate molecules with widely different S values (ex. Nuclei from ribosomes)
2. Sucrose or glycerol gradients or cushions (isokenetic or rate – zonal) – A fixed concentration or a linear gradient of these agents in buffer is used. The compounds increase the density and viscosity of the medium therefore, decreasing the rate at which macromolecule sediment through them and prevening the sedimentation molecules with densities less than the medium. General approach is to pour “cushion” of material at the bottom of the centrifuge tube and centrifuge the virion onto the cushion (cushion need not always be used). By controlling the time and speed of centrifugation a significant purification can be obtained. Since most macromolecules have greater densities than these mediums separation is based on S values. T us can be used to separate molecules with relatively close S values.
3. CsC1 gradient centrifugation (isopycnic or buoyant density) – A linear gradient of these compounds in buffer is prepared in the centrifuge tube. As the concentration of the compound is increased the density of the medium increases in the tube Density is low at the lop am high at the bottom. Macromolecule centrifuged through is ill form a band at a position equal to their buoyant density. Useful for separating
molecules of different densities even when the densities are very close. Drawback is that CsC1 can permanently inactivate some viruses.
Conclusion
Virus can then be separated from materials by differential centrifugation density gradient centrifugation, column chromatography and electrophoresis.
Ultracentrifuge is capable of generating large centrifugal fields by rotating samples at 20,000–100,000 rpm
Summary
§ Viruses can be purified from tissue culture mediums.
§ Virus can then be separated from materials by differential centrifugation, density gradient centrifugation, column chromatography and electrophoresis.
§ Viruses can also be purified by high-speed centrifugation in density gradients of Cesium Chloride (CsC1), potassium tartarate, potassium citrate of sucrose.
Icosahedral viruses are easier to purify than enveloped viruses because enveloped viruses contain variable amounts of envelope per particle, the virus population is heterogeneous in both size amid density.
stain background hut not the – virus particles) or shadowing tech n ‘nines (place specimen a support and direct a vaporized heavy metal across the sample at an angle. This creates a region where relatively link: metal deposits just behind the viral particle (resulting in a shadow).
X – ray crystallography involves the analysis of crystallized virus. Virus crystals are symmetrical structures composed of many isometric viruses. The atoms of the crystal will diffract X – rays in a structure dependent manner. This approach has been used to analyze the structure of the viruses at the molecular level. Resolution at the Armstrong level (10– 10 meters, in the bond length range) is possible.
A purified physical particle should fulfill the following criteria before is identified as a virus panicle:
1. The particle can be obtained only from infected cells or tissues.
2. Particles obtained from various sources are identical regardless of the cellular species in which virus is grown.
3. The degree of infective activity of the preparation vanes directly with the number of particles present.
4. The degree of destruction of the physical particle by chemical or physical means is associated with a corresponding loss of virus activity.
5. Certain properties of the particles and infectivity must be shown to be identical such as their sedimentation behavour in the ultracentrifuge and their PH stability curves
6. The absorption spectrum of the purified physical particle in the ultraviolet range should coincide with ultraviolet inactivation spectrum of the virus.
7. Antisera prepared against the infective virus should react with the characteristic particles and vice versa. Direct observation of an unknown virus can be accomplished by electron microscopic examination fo aggregate fromate in a mixture of antisera and crude virus suspension.
8. The particles should be able to induce the characteristic disease in – vivo (if such experiment are feasible).
9. Passage of the particles in tissue culture should result in the production of progeny with biologic and serologic properties of the virus.
17.3 Preservation of Virus
The preservation of virus is an important sensitive area in virology, it is necessary to preserve viruses after being purified to research purposes and in the development of vaccines. Viruses cannot be preserved on ordinary laboratory media as in most bacteria or fungi. They are preserved as follows:
17.3.1 Freezing
A large wide- mouthed thermo jar or insulated carton, half filled with pieces of solid Co2 (dry ice), serves for transport and storage of material containing viruses. If dry ice is unavailable,
17.3.2 Cultivation and Purification of Viruses
The specimens should be kept co id and transported on ordinary. The temperature in a dry ice storage cabinet is close to -760C. Electric deep freezer can maintain temperatures of – 500C to – 1050C.
17.3.3 Lyophilization
This procedure consists of rapid freezing at lo\v temperature (in a bath containing Alcohol and dry ice) and dehydration from the frozen state at high vacuum: 1.0-50% of normal plasma or serum in the fluid menstruum protects the virus to be frozen and dried. The plasma or serum must not contain neutralizing antibodies. Skimmed milk also another "protective" menstruum in which virus-containing material may he suspended.
17.4 Ethics in a Virology Laboratory
The virology lab is a place where the scientist needs to take special caution in addition to normal laboratory practices:
§ You must wear a sterile laboratory coat every time.
§ You must wear a shoe cover.
§ You mustn't eat in the laboratory.
§ You must not wear make ups, jewelry or wear your hair down in the laboratory.
§ The work benches must be free of unnecessary items such as bags.
§ Nose mask and sterile gloves must be available at all times.
§ You mustn't talk when working with RNA viruses as RNAases are everywhere and may degrade your RNA genome.
§ Safety signs must be in appropriate places and on chemicals.
§ Proper storage of chemicals before and alters use.
§ Proper labeling of samples and chemicals.
§ The laboratory must be clean at all times.