Methods used in Virology

1. LECTURE 3: Methods used in Virology Viro100: Virology 3 Credit hours NUST Centre of Virology & Immunology

2. 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. ATTACHMENT VIRAL LIFE CYCLE HOST FUNCTIONS PENETRATION UNCOATING Transcription Translation REPLICATION ASSEMBLY (MATURATION) RELEASE MULTIPLICATION

4. The Infectious Cycle

5. Some important definitions • Asusceptiblecell has a functional receptor for a given virus, the cell may or may not be able to support viral replication • A resistantcell 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

6. 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.

7. Methods used in Virology • Cultivation of viruses • Isolation of viruses • Centrifugation • Structural investigation of cells and virion • Electrophoretic techniques • Detection of viruses and virus components • Infectivity assays

8. 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)

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

10. Animal Cell Culture

11. Cell Line G= gap

12. 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

13. 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

14. 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

17. 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

18. Partial Purification by Differential Centrifugation

19. Purification of Virion by Density Gradient Centrifugation Rate zonal centrifugation involves layering the preparation on top of a pre-formed gradient. 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.

20. 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

21. Light 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

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