Transcript Isolation, Identification and Cultivation

ISOLATION, QUANTIFICATION
AND identification OF
VIRUSES
REVISION
• The Lytic Cycle
VIRULENT VIRUS
- Upon entering the host, the virus circular
DNA
will undergo multiplication and lyses the host
to release the new virion.
– Culminates in the
death of the host
cell
– Virulent viruses
reproduce only by
lytic cycle.
– T4 virulent phages
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 virusproducing factory, and the cell soon lyses and
releases its viral products.
• 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
• Regardless of the type of virus, the
parasite diverts the host cell’s resources
for viral production.
• The host cell provides:
 Nucleotides for nucleic acid production
 Enzymes
 Ribosomes
Machinery for protein
 tRNA
synthesis
 Amino acids
 ATP
Phage Growth
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
Lesson Outcome
• Explain the cultivation and quantification techniques
for bacteriophages
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.
Cultivation and identification of phages
1. Obtaining bacteriophage from sample
2. Amplification/multiplication of phages
Solution (sample) into liquid media (eg. NB,
increase the numbers of phages
TSB)
(in the sewage sample) by
Addition of host – sewage: enteric bacteria, faeces:
allowing them to infect and
E.coli
reproduce
Incubation: 37o C, 24
within fresh host.
hrs
1. Isolation of multiplied phages
Separate the remaining host cell/cell debris
via centrifugation and filtration (0.2µm
filter)
1. Plaque assay
Preparation of pure phage
suspension
Detection, identification , phage isolation for storage and
future research
Isolation and identification of phages –
Plaque assay technique
 Plaque assay technique
 STEPS:
Detection, isolation,
identification, characterisation of
phages
1. Serial dilutions –ten-fold dilution in preparation of phage
suspension
1.
2.
3.
4.
5.
Add in host (log-phase growth) to phage dilution
Incubation 37o C, 20 min
Add in top agar
Pour on solidified agar
Incubate 37o C, 18-24 hrs.
To allow infection of
phage to host
Isolation and identification of phages –
Plaque assay technique
 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 37o C, 20 min
4. Add in top agar
5. Pour on solidified agar
6. Incubate 37o C, 18-24 hrs.
7. Observation of plaque
formation
Plaque: can be collected for
storage
Identification of phages –
Plaque assay technique
 Plaque ?
Zone of cell death/ a clear area in a bacterial lawn culture where
viruses have lysed host cells
HOW TO
IDENTIFY
TEMPERATE- Cloudy
plaque
PHAGE?
 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
 Basis of plaque formation:
 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
QUIZ
1. List the replication steps for animal viruses.
Adsorption, Penetration, Uncoating, Synthesis, Maturation,
Release
2. Name the point of entry and exit for animal viruses.
Entry: endocytosis and fusion of virus envelope to host cell
membrane
Exit: budding/exocytosis and lysis
3. Name the site for replication, protein synthesis and
maturation step for DNA virus.
Replication: nucleus Protein synthesis: cytoplasm Maturation:
nucleus
4. Define “plaque”.
A clear area in a bacterial lawn culture where viruses have lyzed host
cells
5. How do you identify the present of lambda phage
through plaque assay technique?
Formation of cloudy/not clear plaque because lambda phage is temperate
phage
ISOLATION, QUANTIFICATION
AND identification OF
VIRUSES
Overview of Animal Viruses
Overview of animal
virus actions
-
Lesson Outcome
• Explain the cultivation and quantification techniques
for animal viruses
Isolation, Cultivation and Identification of animal
viruses
1. In living animals
CULTIVATION/ ISOLATION
- 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 system’s response
to viral infections.
- HIV: immunodeficient mice grafted to produce human T cells and
human gamma globulin.
IDENTIFICATION
- 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
Isolation, Cultivation and Identification of animal
viruses
2. In Embryoted egg
CULTIVATION/ ISOLATION
- use embryonated chicken, duck or
turkey for inoculation of viral
suspension.
IDENTIFICATION
- 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
Viral culture in eggs: Some viruses, such as influenza viruses,
are grown in embryonated chicken eggs
3. Using cell culture
CULTIVATION/ ISOLATION
- 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.
IDENTIFICATION
- 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
Culturing of using cell culture
•
1.
2.
•
Two discoveries greatly enhanced the usefulness of cell
cultures for virologists and scientists
The discovery and use of antibiotics made it possible to
prevent bacterial contamination
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
Identification of viruses
1. PCR – polymerase chain reaction
2. Restriction fragments
polymorphisms (RFLP)
3. Serological method – Western blot
common method use
4. Immunological test , ELISA,
agglutination test – if specific
preparation of killed, inactivated
antibody is Aavailable
or attenuated microorganisms to
induce artificially acquired active
immunity
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
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