Introduction to Lab Ex. 21: Bacteriophages

1. Introduction to Lab Ex. 21: Bacteriophages

2. Viruses are characterized from all other organisms by specific features. They are not considered as truly living organisms due to their inability to reproduce outside of a living cell. Characteristics of all viruses include: ·Obligate intracellular parasites ·        Filterable structures – very small ·        Consist of outer protein coat and inner genome ·        Contain only one l kind of nucleic acid – either RNA or DNA ·        Lack independent metabolic abilities Viruses are classified based on their structure and the kind of hosts they infect: RNA/ DNA viruses; animal/plant/bacterial viruses. We will be working with bacterial viruses since they are easier to grow and study in the lab. Bacterial viruses are called bacteriophages since (“phage” meaning “eating cell”) the bacterial host cells appear to be devoured by the viruses.

3. Bacteriophages are named using the alphanumerical system: use an alphabet and a number to name them; example T1 phage, T2 phage etc. Viruses may also have one of two shapes associated with their structure: they may be helical or icosahedral. Bacteriophages may be tailless, tailed or filamentous. The structures of phages include: the outer capsid that is made of protein (individual units called capsomeres) and an inner genome containing the nucleic acid. The nucleic acid may be either RNA/DNA never both and it may be either single stranded or double stranded.

4. Viruses need to get into their host cell on order to replicate (multiply/reproduce). While they can remain unchanged in the environment, they need living hosts for replications processes since they have no independent ability to metabolize and generate energy. They are able to take over the host cell and direct the cell into viral replicative processes.

5. Virus infections of host cells can lead to one of two results: lysis or lysogeny. In the lytic cycle the infected host cell is lysed soon after infection starts. In the lysogenic cycle host cell lysis does not usually follow infection immediately – the viral nucleic acid attaches to the host cell genome and can remain dormant for indefinite periods of time without continuing the normal stages of a viral infection as seen in the lytic cycle.

6. The lytic cycle of viral infection of a host cell can be described in 4 stages: ·        Adsorption ·        Penetration ·        Intracellular biosynthesis of viral products ·        Release Adsorption is the phase wherein the virus attaches to the surface of its host cell. This is mainly through the attachment proteins on the virus surface that interact with surface receptors on the cell’s surface. Thus this phase of the infection is specific for the virus – it can only infect those cells that have the complimentary receptor sites – host specificity. Also, since the attachment proteins are mostly proteins, it also makes the viruses sensitive to environmental factors such as heat – heat sensitivity.

7. Viruses first need to attach to the host cell surface before the infection proceeds to the next stage. Penetration is the process where the viral genome/nucleic acid is injected/enters the host cell cytoplasm. The viral capsid stays outside the host cell and only the nucleic acid is found within the host cell. Intracellular biosynthesis of viral products then follows where the viral nucleic acid directs the synthesis of progeny viral proteins and nucleic acid molecules using the cells energy and raw materials. Once the viral products have been synthesized, they are then assembled into progeny viral particles. Release is the phase of infection where the new viral particles are released out of the host cell either through excretion methods or through lysis of the host cell.

8. Viruses are quantitated based on the effects they have on host cells especially those that result in the lysis of the host cell. One method is the Plaque assay. Plaque assay relies on the fact that one host cell is infected by one virus resulting in its lysis. Bacteriophage plaque assays are relatively simple procedures to quantitate viruses. The host cell culture is mixed with a dilution of the virus suspension and the mixture added on the surface of culture media plates. As the host cells grow and form a lawn of growth on the plate surface the plaques arising out of the lysis of the host cells due to the viral infection become visible to the eye and can be counted to quantitate the viruses in the suspension.

9. Thus a plaque is a clearing in a lawn of bacterial growth caused by the repeated lysis of the host cells as a result of continued viral infection.