ISOLATION, QUANTIFICATION AND identification OF VIRUSES
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REVISION The Lytic Cycle Culminates in the death of the host
cell Virulent viruses reproduce only by lytic cycle. T4 virulent
phages The Lysogenic Cycle Replication of the viral genome without
destroying the host cell. A temperate virus may reproduce by either
cycle. Lambda virus (temperate phage): resembles T4 but only has a
single short tail fiber TEMPERATE VIRUS -Within the host, the virus
circular DNA engages in either the lytic or lysogenic cycle. -
During a lytic cycle, the viral genes immediately turn the host
cell into a virus- producing factory, and the cell soon lyses and
releases its viral products. VIRULENT VIRUS - Upon entering the
host, the virus circular DNA will undergo multiplication and lyses
the host to release the new virion.
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Regardless of the type of virus, the parasite diverts the host
cells resources for viral production. The host cell provides:
Nucleotides for nucleic acid production Enzymes Ribosomes tRNA
Amino acids ATP Machinery for protein synthesis
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Growth curve for a bacteriophage: The eclipse phage represents
the time after penetration through the biosynthesis of mature
phages. The latent period represents the time after penetration
through release of mature phages. The number of viruses per
infected cell is the viral yield, or burst size Phage Growth
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Lesson Outcome Explain the cultivation and quantification
techniques for bacteriophages
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Cultivation and identification of viruses The primary purposes
of viral cultivation are: 1. to isolate and identify viruses in
clinical specimens 2. to prepare viruses for vaccines 3. to do
detailed research on viral structure, multiplication cycles,
genetics, and effects on host cells. Bacteriophages cultivation and
identification is simple and easy, due to the simplicity of the
host cells. Animal viruses difficult, due to the properties of the
animal host. Systems of cultivation with broader applications were
developed, including in vitro* cell (or tissue) culture methods and
in vivo* inoculation of laboratory-bred animals and embryonic bird
tissues. - Such use of substitute host systems permits greater
control, uniformity, and wide-scale harvesting of viruses.
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Cultivation and identification of phages 1.Obtaining
bacteriophage from sample 2.Amplification/multiplication of phages
1.Isolation of multiplied phages 1.Plaque assay Solution (sample)
into liquid media (eg. NB, TSB) Addition of host sewage: enteric
bacteria, faeces: E.coli Incubation: 37 o C, 24 hrs Separate the
remaining host cell/cell debris via centrifugation and filtration
(0.2m filter) Preparation of pure phage suspension Detection,
identification, phage isolation for storage and future research
increase the numbers of phages (in the sewage sample) by allowing
them to infect and reproduce within fresh host.
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Plaque assay technique STEPS: 1.Serial dilutions ten-fold
dilution in preparation of phage suspension 1.Add in host
(log-phase growth) to phage dilution 2.Incubation 37 o C, 20 min
3.Add in top agar 4.Pour on solidified agar 5.Incubate 37 o C,
18-24 hrs. 6.Observation of plaque formation Isolation and
identification of phages Plaque assay technique Detection,
isolation, identification, characterisation of phages To allow
infection of phage to host
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Plaque assay technique STEPS: 1.Serial dilutions ten-fold
dilution in preparation of phage suspension 2.Add in host
(log-phase growth) to phage dilution 3.Incubation 37 o C, 20 min
4.Add in top agar 5.Pour on solidified agar 6.Incubate 37 o C,
18-24 hrs. 7.Observation of plaque formation Isolation and
identification of phages Plaque assay technique Plaque: can be
collected for storage
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Plaque ? The basis is that one viral particle infects one cell,
is replicated and the cell lyses. The nearby cells are infected and
a plaque of dead cells is formed over time. Identification of
phages Plaque assay technique Zone of cell death/ a clear area in a
bacterial lawn culture where viruses have lysed host cells HOW TO
IDENTIFY TEMPERATE PHAGE? - Cloudy plaque
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Basis of plaque formation: Identification of phages Plaque
assay technique Plaque assay also to calculate number of phages
present. The titer of a phage suspension, is determined by counting
the number of plaques that form from a given volume of suspension.
Phage titer is expressed as plaque forming units (PFU) per
milliliter (ml). pfu/ml * measurement of the number of viable,
infectious bacteriophage
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QUIZ 1. List the replication steps for animal viruses. 2. Name
the point of entry and exit for animal viruses. 3. Name the site
for replication, protein synthesis and maturation step for DNA
virus. 4. Define plaque. 5. How do you identify the present of
lambda phage through plaque assay technique? Adsorption,
Penetration, Uncoating, Synthesis, Maturation, Release Entry:
endocytosis and fusion of virus envelope to host cell membrane
Exit: budding/exocytosis and lysis Replication: nucleus Protein
synthesis: cytoplasm Maturation: nucleus A clear area in a
bacterial lawn culture where viruses have lyzed host cells
Formation of cloudy/not clear plaque because lambda phage is
temperate pha ge
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ISOLATION, QUANTIFICATION AND identification OF VIRUSES
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Overview of Animal Viruses - Overview of animal virus
actions
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Lesson Outcome Explain the cultivation and quantification
techniques for animal viruses
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Isolation, Cultivation and Identification of animal viruses 1.
In living animals - using live animal eg.mice, rats, rabbits,
guinea pigs, hamster, chickens, and monkey. - the animal is exposed
to the virus by injection of a viral preparation or specimen into
the brain, blood, muscle, body cavity, skin, or footpads. - use in
example research to study the immune systems response to viral
infections. - HIV: immunodeficient mice grafted to produce human T
cells and human gamma globulin. - The signs of viral growth include
death of the animal and defects in animal development. The infected
animal tissue can be prepared for examination with an electron
microscope CULTIVATION/ ISOLATION IDENTIFICATION
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Isolation, Cultivation and Identification of animal viruses 2.
In Embryoted egg - use embryonated chicken, duck or turkey for
inoculation of viral suspension. - The signs of viral growth
include death of the embryo, defects in embryonic development, and
localized areas of damage in the membranes, resulting in discrete,
opaque spots called pocks (a variant of pox). The embryonic fluid
and tissue can be prepared for examination with an electron
microscope. - Some can also be detected by their ability to
agglutinate red blood cells or by their reaction with an antibody
of known specificity that will affix to its corresponding virus, if
it is present. IDENTIFICATION CULTIVATION/ ISOLATION
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Viral culture in eggs: Some viruses, such as influenza viruses,
are grown in embryonated chicken eggs
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3. Using cell culture - preferred type of growth medium for
virus, more convenient than the previous two methods - use isolated
cell from animal that are cultured invitro. Normal cells will form
monolayer. - If viruses are present, the cells of monolayer will
deteriorate as they multiply. Cell deterioration is called
cytopathic effect (CPE). CPE can be detected and counted = plaques
by phages (plaque assay). Microscopic observation via electron
microscope (histopathology). A. Normal B. Transformed CULTIVATION/
ISOLATION IDENTIFICATION
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Culturing of using cell culture Two discoveries greatly
enhanced the usefulness of cell cultures for virologists and
scientists 1.The discovery and use of antibiotics made it possible
to prevent bacterial contamination 2.The discovery of proteolytic
enzymes (e.g. trypsin) can free animal cells from surrounding
tissues without injuring freed cells Subculturing: the process by
which cells from an existing culture are transferred to new
containers with fresh nutrient media
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Identification of viruses 1. PCR polymerase chain reaction 2.
Restriction fragments polymorphisms (RFLP) Serological 3.
Serological method Western blot common method use Immunological 4.
Immunological test, ELISA, agglutination test if specific antibody
is available Vaccine development 1.Embryoted chicken egg one the
most used method of viral isolation and growth 2.Still used to grow
viruses for some vaccines eg. Influenza vaccine 3.Cell culture and
animal tissue are also used in vaccine preparation for some
viruses. A preparation of killed, inactivated or attenuated
microorganisms to induce artificially acquired active immunity
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QUESTION Briefly explain the culturing method used to identify,
isolate and cultivate animal viruses. Embryonated eggs : use
embryonated chicken, duck or turkey for inoculation of viral
suspension. The signs of viral growth include death of the embryo,
defects in embryonic development, and localized areas of damage in
the membranes, resulting in discrete, opaque spots called pocks (a
variant of pox). The embryonic fluid and tissue can be prepared for
examination with an electron microscope. Tissue culture: use
isolated cell from animal or plant that are cultured invitro. The
cells will form monolayer. Thesign of viral growth detected through
formation of plaque or looking at cytopathic effect. Animal : using
live animal eg. mice, rats, rabbits, guinea pigs, hamster,
chickens, and monkey. The signs of viral growth include death of
the animal and defects in animal development. The infected animal
tissue can be prepared for examination with an electron microscope.
- Identification: also by PCR, serology