Introduction to Lab Ex. 21: Bacteriophages

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Introduction to Lab Ex. 21 Bacteriophages
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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.
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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.
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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.
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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.
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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 cells
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.
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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.  
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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.
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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.