Cultivation of Viruses, Biology tutorial

Detection of virus infected cells:

Multiplication of viruses can be monitored in variety of ways some of which are:

1. Cytopathic Effects (CPE) Growth of viruses in cells causes morphological changes in cells. Kinds of virus in cells or necrosis, cytoplasmic vacuolization. Most viruses generate some obvious CPE comprises lysis or necrosis, cytoplasmic vacuolization. Most viruses generate obvious CPE in infected cells which is usually characteristic of virus group.

2. Appearance of the virus-coded protein, like haemagglutinin of influenza virus. Specific antisera can he utilized to detect synthesis of viral proteins in infected cells.

3. Adsorption of erythrocytes to infected cells known as haemasorption, because of the presence of virus-encoded haemagglutinin (Parainfluenza and influenza) in cellular membranes. This reaction becomes positive prior to cytopathic changes are visible and in some cases happen in absence of CPE.

4. Interference by the non-cytopathogenic virus (like rubella) with replication and induction of CPE by the second challenge virus (like echovirus) added as the indicator

5. Morphologic transformation by the oncogenic virus (like Raus Sarcoma virus), generally accompanied by loss of contact inhibition and piling up of cells in discrete foci.

6. Virus growth in the embryonated chick egg result in death of embryo (like encephalitis virus), production of pocks or plaques on CAM, (like herpes, small pox vaccina), development of haemagglutinin in embryonic fluids or tissues (like influenza) or development of infective virus (like Poliovirus types 2).

Cultivation:

Several viruses can be grown in cell cultures or in fertile eggs (embryonated eggs) under severely controlled conditions or inoculation in appropriate host animals.

Embryonated eggs:

These are more practical for cultivation of viruses, ethical and economic and [or ease of handling and relative freedom from contaminants. A developing chick embryos are immune-deficient therefore favors growth of viruses. Many viruses will grow or can be adapted to grow fertile eggs, and few may kill embryo or may generate visible proof of specific infection on choricallantoic membrane (C.A.M). Haemagglutinating viruses in allantioc and amniotic fluids will cause haemagglutination when incubated with suitable species of erythrocytes (red blood cells). There are 4 routes of inoculating eggs for viral cultivation:

  • C.A.M utilized for several pox and some herpesviruses.
  • Allantoic cavity utilized for ortho- paramyxo and rhabdo -viruses.
  • Amniotic cavity utilized for ortho-and paramyxo-viruses.
  • Yolk sac utilized for many togaviruses

Animal host:

Preferably, natural host of the viruses or closely related species must be utilized for animal inoculation. This mode of viral cultivation is not always practical on ethical or economic ground, whereas there is also the option of latent infection with virus under consideration.

Route of inoculation of animal is the significant factor because of the specific affinity some viruses have for definite tissues. Like lntracerebral- inoculation of mice with rabies virus; subcutaneous inoculation of swine vesicular disease virus in pigs. Animal may illustrate clinical signs of infection and these should be observed, or biopsy material taken for examination. Neutralization of virus with the specific antiserum before inoculation of animals will, certainly prevent occurrences of infection. Growth of virus in animal hosts is still utilized for main isolation of certain viruses and for studies of pathogenesis of viral disease and of viral oncogenesis.

Cell cultures:

Availability of cell grown in-vitro has made easy the recognition and cultivation of newly isolated viruses and classification of previously known ones. Cell cultures have been on success as advent of antibiotics and fungicides that have made it possible to prevent contamination of cultures. Introduction of trypsin facilitated monolayer growth of cells. There are three basic kinds of cell culture:

(a) Primary cultures are made dispersing cells (generally with trypsin) from freshly removed host tissues. Generally, they are not able to grow for more than a few passages in culture like Monkey Kidney cells and Human-amnion cells.

(b) Secondary cultures (semi-continuous cells) are also called as Diploid cells. They have gone through a change which permit their limited cultures (up to 50 passages) but which keep their normal chromosome pattern like Human embryo lung.

(c) Continuous cell lines are cultures able to more prolonged, maybe indefinite growth which have been derived from diploid cell lines or create malignant tissues. They regularly have altered and irregular numbers of chromosomes. The kinds of cell culture utilized for virus cultivation depend on sensitivity of the cells to the particular virus. Continuous cell Lines is also referred to as Heteroploids cells lines like Hela cells derived from human cervical cancer.

Purification of viral particles:

For purification, starting material is generally large volumes of tissue culture medium, body fluids or infected cells. Pure virus is significant so as to have significant studies on properties and molecular biology of virion. The first often involved concentration of virus particles by precipitation with ammonium sulphate, ethanol or polyethylene glycol or by ultra filtration. Haemagglutination and elution can be utilized to concentrate orthomyxoviruses. Once concentrated, virus can then be divided from materials by differential centrifugation, density gradient centrifugation, column chromatography and electrophoresis. More than one step is generally essential to attain sufficient concentration. The preliminary purification will eliminate non-virus material: First step may comprise centrifugation while final purification step roughly always involves density gradient centrifugation. Band of purified virus may be identified by optical methods, by following radioactivity if virus is radiolabeled, or by examining infectivity. Viruses can be purified by high speed centrifugation in density gradients of Cesium Chloride (CsCl), potassium tartrate, potassium citrate of Sucrose. Gradient material of choice is the one that is slightest toxic to the virus. Virus particles migrate to equilibrium position where the density of the solution is equivalent to buoyant density and create visible band, Virus bands are harvested by puncture through bottom of plastic centrifuge tube and examined for infectivity.

Centrifugation:

Centrifugation as the purification and characterization procedure:

Ultracentrifuge: The centrifuge is able to generate large centrifugal fields by rotating samples at 20,000-100,000 rpm. Centrifugal forces of greater than 100,000 X gravity can be produced.

Sedimentation coefficient:

  • Rate at which macromolecule sediments under the defined gravitational force.
  • This parameter is affected by both molecular weight and shape of the macromolecule (larger and more spherical sed. faster).
  • The basic unit is Svedberg (S) that is 10-13 sec.
  • This value can be utilized to approximate molecular weights in conjunction with other values.

Buoyant density: Density at which virus or other macromolecule neither sinks nor floats when suspended in the density gradient (like, CsCl2 or sucrose).

Types of sedimentation medium:

1. Aqueous Buffer (Water based): Can be utilized to separate molecules with extensively different values (e.g. Nuclei from ribosomes)

2. Sucrose or glycerol gradients or cushions (isokenetic or rate-zonal): Fixed concentration or the linear gradient of the agents in buffer is utilized. Compounds increase density and viscosity of medium thus, decreasing rate at which macromolecule sediment through them and preventing sedimentation molecules with densities less than medium. General approach is to pour the cushion of material at bottom of centrifuge tube and centrifuge the virion on cushion (cushion need not always be utilized). By controlling time and speed of centrifugation the important purification can be attained.

3. CsCl gradient centrifugation (isopycnic or buoyant density): The linear gradient of these compounds in buffer is made in centrifuge tube. As concentration of compound is increased density of medium also increases in tube. Density is low at the top and high at the bottom. Macromolecule centrifuged through will create the band at the position equivalent to their buoyant density. Helpful for dividing molecules of different densities even when densities are very close. Disadvantage is that CsCl can enduringly inactivate some viruses. Evaluating purity of virions and recognition of the viral particle

Methods for assessing the purity of virions:

i) Spectrophotometric analysis: UV absorption at 260 and 280 nm. This ratio (260/280) is the characteristic of the pure virus and is dependent on amount of nucleic acid and protein in virus. Number can be utilized to evaluate amount in preparation. Nucleic acid absorbs light approx twice as well at 260 vs. 280 and vice versa for protein.

ii) Serological methods: Antibodies to viral proteins are utilized to distinguish, identify, or quantify virions. Antibodies can be made in numerous ways. Entire virus (possibly attenuated (modified so cannot cause disease) can be injected in animals (rabbit or mouse) and monoclonal (single kind of antibody generally identifies single epitope) or polyclonal (several different antibodies which may identify numerous epitopes).

iii) Electron microscopy Method: It permits visualization of single virus particles. It is based on principle of electron scattering. The beam of electrons is focused on sample. Electrons inside specimen will scatter electron beam. Scattering effect is improved by presence of heavy, electron rich metal ions (that is gold, platinum) inside the sample.

iv) X-ray crystallography: It involves examination of crystallized virus. Virus crystals are symmetrical structures made up of several isometric viruses. Atoms of crystal will diffract X-rays in the structure dependent manner. This approach has been utilized to examine structure of viruses at molecular level. Resolution at the Armstrong level (10-10 meters, in bond length range) is possible.

Identification of a viral particle:

The purified physical particle must fulfill given criteria before it is recognized as the virus particle:

1. Particle can be attained only from infected cells or tissues.

2. Particles attained from different sources are identical, in spite of cellular species in which virus is grown.

3. Degree of infective activity of preparation differs directly with number of particles present.

4. Degree of destruction of physical particle by chemical or physical means is related with the corresponding loss of virus activity.

5. Certain properties of particles and infectivity should be shown to be the same, like their sedimentation behavior in ultracentrifuge and their PH stability curves.

6. Absorption spectrum of purified physical particle in ultraviolet range must coincide with ultraviolet inactivation spectrum of virus.

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