Methods used in Virology

LECTURE 3:

Viro100: Virology3 Credit hoursNUST Centre of Virology & Immunology

Virus Discovery

• 1901 first human virus, Yellow fever virus• 1903 Rabies virus• 1906 Variola virus• 1908 chicken leukemia virus, poliovirus• 1911 Rous sarcoma virus• 1915 Bacteriophages• 1933 Influenza virus

3

VIRAL LIFE CYCLE

ATTACHMENT

PENETRATION HOSTFUNCTIONS

ASSEMBLY(MATURATION)

Transcription

REPLICATION

RELEASE

UNCOATING

Translation

MULTIPLICATION

The

Infe

ctiou

s Cyc

le

Some important definitions• A susceptible cell has a functional receptor for

a given virus, the cell may or may not be able to support viral replication

• A resistant cell has no receptor, it may or may not be competent to support viral replication

• A permissive cell has the capacity to replicate virus, it may or may not be susceptible

• A susceptible AND permissive cell is the only cell that can take up a virus particle and replicate it

Methods used in Virology

• Virology is a huge subject and uses a wide range of methods

• Many of the techniques of molecular biology and cell biology are used

• A number of techniques are used, not only in virus research, but also in the diagnosis of virus diseases of humans, animals and plants.

1. Cultivation of viruses2. Isolation of viruses3. Centrifugation4. Structural investigation of cells and virion5. Electrophoretic techniques6. Detection of viruses and virus components7. Infectivity assays

Methods used in Virology

1- Cultivation of viruses

• For the study of virus, we need to produce the object in reasonable amount

• Virus cultivation is also referred to as propagation or growth

• A few techniques have been developed for the cultivation of viruses in cell free systems (data is very poor)

• Appropriate host cells are required in which it can replicate (susceptible AND permissive)

• Plant viruses may be supplied with specially cultivated plants or with cultures of protoplasts (plant cells from which the cell wall has been removed)

• Phages are supplied with bacterial cultures• while animal viruses may be supplied with

whole organisms, such as mice, eggs containing chick embryos or insect larvae

• Animal viruses are grown in cultured animal cells

Animal Cell Culture

G= gap

Cell Line

Most types of cell taken from the body do not grow well in culture. If cells from a primary culture can be sub cultured they are growing as a cell line. They can be sub cultured only a finite number of times unless they are immortalized, in which case they can be sub cultured indefinitely as a continuous cell line. Cancer cells are already immortalized, and continuous cell lines may be established from these without further treatment

• Continuous cell lines consist of cells that have been immortalized, either in the laboratory or in the body,

• They can be subculture indefinitely, Cells are cultured in media that provide– Nutrients– Animal serum– Maintenance of optimum osmotic pressure– pH for the cells– High concentration of carbon dioxide

• Contamination with bacteria and fungi can cause major problems in cell culture work

2. Isolation of viruses

• Many viruses can be isolated as a result of their ability to form discrete visible zones (plaques) in layers of host cells.

• Plaque is formed when infection spreads radially from an infected cell to surrounding cells

• Phages form clear plaques on bacterial lawn• All viruses produced from the virus in the plaque

should be clone, it should be genetically identical• This clone is referred to as isolate, and if this

isolate is distinct from other isolate, then it is called strain

3. Centrifugation• After a virus has been propagated it is usually

necessary to remove host cell debris and other contaminants before the virus particles can be used for laboratory studies, for incorporation into a vaccine, or for some other purpose.

• Many virus purification procedures involve centrifugation; partial purification can be achieved by differential centrifugation and a higher degree of purity can be achieved by some form of density gradient centrifugation

Partial Purification by Differential Centrifugation

Purification of Virion by Density Gradient Centrifugation

Rate zonal centrifugation involves layering the preparation on top of a pre-formedgradient. Equilibrium centrifugation can often be done starting with a suspension of the impure virus in a solution of the gradient material; the gradient is formed during

centrifugation.

4. Structural investigation of cells and virion

• Light Microscopy• Electron Microscopy

• Transmission Electron Microscopy• Scanning Electron Microscopy

• X-ray crystallography• Nuclear Magnetic Resonance• Atomic Force Microscopy

• Virions are beyond the limits of resolution of light microscopes

• Light microscopy has useful applications in detecting virus infected cells

• Cytopathic effects• Confocal microscopy• The principle of this technique is the use of a

pinhole to exclude light from out of focus regions of the specimen.

• Laser

Light Microscopy

Thank You!