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Basic Techniques to Grow Viruses and Study Virus-Host Interactions

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Basic Techniques to Grow Viruses and Study Virus-Host Interactions Growth of Viruses While it is easy to grow bacterial viruses, it is much more difficult and ... – PowerPoint PPT presentation

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Title: Basic Techniques to Grow Viruses and Study Virus-Host Interactions


1
Basic Techniques to Grow Viruses and Study
Virus-Host Interactions
2
Growth of Viruses
  • While it is easy to grow bacterial viruses, it is
    much more difficult and expensive to grow animal
    viruses
  • Whole animals
  • Embryonating eggs (the classic host for vaccine
    production)

3
Growth of Viruses, continued
  • Organ culture - pieces of brain, gut, or trachea,
    etc. containing different cell types are grown in
    culture

4
Organ cultures
Sections through tracheal organ cultures (a)
uninfected (b) infected with a rhinovirus for 36
hours. Note the disorganization of the ciliated
cells (uppermost layer) after infection.
5
Growth of Viruses, continued
  • Cell or tissue culture this is where tissues
    are removed from an organism and are grown in
    vitro, usually in flasks
  • Primary cultures are cells that have been
    directly derived from a tissue and placed in
    culture.
  • Are differentiated
  • They, like the tissue from which they were
    derived, have a limited life span.
  • Most will grow attached to the flask as a
    monolayer of cells one cell thick.

6
Making a primary cell line
7
Growth of Viruses, continued
  • Cell lines
  • Are dedifferentiated
  • Are diploid
  • Survive more passages than primary cell lines,
    but eventually die
  • Immortalized cells or continuous cell lines are
    cells that have a mutation or mutations that
    allow the cells to be passaged many times, i.e.
    they dont have a limited life span.
  • Are usually heteroploid
  • Most were originally derived from a tumor.
  • Most grow as monolayers, though a few grow in
    suspension.

8
Making a continuous cell line
9
Tissue Culture Cells
10
Growth of Viruses, continued
  • When cells grow as monolayers, they can be used
    to quantify the number of animal viruses using a
    plaque assay.
  • The virus is serially diluted in a liquid medium.
  • For each dilution a set amount is added to a
    separate plate containing a monolayer of tissue
    culture cells and the viruses in that solution
    are allowed to attach to the tissue culture
    cells.
  • After attachment has been allowed to occur, a
    semi-solid medium is added to restrict the
    movement of new viruses produced so that only
    adjacent cells will be infected.

11
Growth of Viruses, continued
  • Where virus has infected the tissue culture
    cells, the infected cells will die causing the
    formation of a clear zone amongst the otherwise
    intact monolayer of cells
  • This clear zone is called a plaque and it
    theoretically represents an area where one virus
    has infected a single tissue culture cell, has
    multiplied and been released, and has gone on to
    infect adjacent cells.
  • The number of plaque forming units (pfu)/ml can
    be calculated based on the dilution of the
    original viral solution.
  • The term pfu/ml is used rather than the number of
    viruses/ml because it is possible that
    occasionally more than one virus infects a single
    cell.
  • Often the cells or plaques are stained to help
    in visualization of the plaques.

12
Serial dilutions
13
Animal Virus Plaque Assay
14
Plaque assay results
15
Basic Techniques to Study Viruses and Virus-Host
Cell Interactions
  • Serological and immunological methods these
    tests are often used for diagnosis of viral
    infections
  • May assay directly for the virus (direct assay)
  • May assay for antibodies, produced in the host,
    against the virus (indirect assay)
  • Hemagglutination assay-a direct method to titer
    virus.
  • Is based on the ability of some viruses to
    agglutinate RBCs.
  • Virus is titered by making serial two-fold
    dilutions of the virus and determining the
    highest dilution of virus that causes
    agglutination of the RBCs.

16
Hemagglutination assay
17
Hemagglutination assay
18
Serological/Immunological Methods
  • Hemagglutination-Inhibition Assay an indirect
    test for antibody against specific viruses that
    can agglutinate RBCs.
  • Mix serial dilutions of patients sera with the
    virus that is the suspected causative agent of
    the patients infection, and then add RBCs.
  • If the patient has antibodies specific to the
    virus, they will bind to the virus and prevent
    the virus from agglutinating the RBCs.

19
Hemagglutination inhibition assay
20
Serological/Immunological Methods
  • Immunofluorescence may be either
  • direct and test for the presence of viral
    antigen in tissues or
  • indirect and test for the presence of antibodies
    against a specific virus in a patients sera.
  • This method uses an antibody with a fluorescent
    tag attached to it.
  • With the direct test, the antibody that is tagged
    is an antibody against the virus that one is
    testing for.
  • In the indirect test, the tagged antibody is an
    antibody against another antibody, i.e.
    anti-human IgG. The presence of the fluorescent
    tag is detected by looking under a fluorescent
    microscope.

21
Direct immunofluorescent antibody test
22
Indirect immunofluorescent antibody test
23
Immunofluorescence
?
?
24
Serological/Immunological Methods
  • ELISA (enzyme linked immunosorbent assay)
  • Can either be direct (tests for virus) or
    indirect (tests for antibody to virus).
  • ELISA is similar to the immunofluorescent assays,
    but differs in the type of molecule that is
    tagged to the antibodies that are used.
  • The molecule that is attached to an antibody in
    an ELISA assay is an enzyme.
  • The presence of the enzyme is detected by adding
    a substrate to the enzyme which when acted upon
    by the enzyme produces a colored product.
  • An indirect ELISA test is used to screen
    individuals for HIV infection.

25
Direct (sandwich) ELISA
(virus?)
26
Indirect ELISA
virus
against virus?
27
Indirect versus direct (sandwich) ELISA
28
ELISA (sandwich method to detect Ag)
29
ELISA (indirect)
30
Serological/Immunological Methods
  • Western immunoblot-
  • A Western immunoblot can be either direct or
    indirect.
  • The Western immunoblot analyzes a sample for a
    specific protein(s) (direct) or for antibodies
    against a specific protein(s) (indirect).
  • The screening test to diagnose HIV is the
    indirect ELISA test.
  • The indirect Western immunoblot is used to
    confirm a positive ELISA test.

31
Western immunoblot
32
Western Blot
33
Indirect Western immunoblot for HIV diagnosis
34
Indirect Western immunoblot for HIV diagnosis
35
Basic Techniques to Study Viruses and Virus-Host
Cell Interactions
  • Ultrastructural studies used for purification
    purposes
  • Physical methods
  • Size by filtration- molecular sieve
    chromatography. Uses a column filled with beads
    containing holes.
  • Large molecules are excluded from the holes and
    come off the column first.
  • Small molecules enter the holes in the beads and
    therefore move slower down the column, coming off
    the column after large molecules.

36
Molecular Sieve Chromatography
37
Physical methods
  • Centrifugation
  • Can pellet materials (virus) by centrifugation
  • Equilibrium density gradient centrifugation an
    inert material is used and it forms a density
    gradient during the centrifugation. Materials
    (virus) are forced down until they reach a
    density that buoys them up.
  • Rate-zonal centrifugation similar to density
    gradient centrifugation, but uses a preformed
    gradient rather than generating a gradient during
    the centrifugation process.

38
Centrifugation
39
Equilibrium density gradient centrifugation
40
Physical methods
  • Electrophoresis materials are forced through a
    meshwork of matrix material (agarose or
    polyacrylamide) by an electric current.
  • Usually used for nucleic acids or proteins which
    are separated on the basis of size, shape, and
    charge.

41
Electrophoresis
42
Physical methods
  • Affinity chromatography Takes advantage of
    highly specific binding interactions.
  • A column is made with a material that has a
    specific receptor (binding interaction) for the
    substance you are trying to purify (for example
    the receptor for a particular virus).
  • A solution from which you wish to purify your
    virus is run through the column.
  • The virus binds to the receptor, but everything
    else is washed through the column.
  • Next you run a new solution through the column
    which changes the conditions (pH, ionic strength,
    etc.) in the column to those in which the
    specific virus-receptor interaction no longer
    occurs.
  • The virus will be eluted from the column.

43
Affinity chromatography
44
Physical methods
  • X-ray crystallography
  • Chemical methods to determine the overall
    composition and the nature of the nucleic acid
  • Electron microscopy
  • Whole mounts
  • staining (heavy metals)
  • - staining
  • Ultrathin sections

45
Basic Techniques to Study Viruses and Virus-Host
Cell Interactions
  • Molecular biology often used to study the
    structure of the nucleic acid
  • Hybridization to come together through
    complementary base-pairing.
  • Can be used in identification.
  • For in situ (or plaque) hybridization the tissue
    containing the putative organism is treated to
    release the nucleic acid which is then denatured
    to single strands.
  • Labeled single-stranded DNA (a probe) unique to
    the organism you are testing for is added and
    hybridization is allowed to occur.
  • Unbound probe is washed away and the presence of
    bound probe is determined by the presence of the
    label.

46
In situ hybridization
47
Molecular Biology
  • Polymerase chain reaction used to amplify
    something found in such small amounts that
    without PCR it would be undetectable.
  • Uses two primers, one that binds to one strand of
    a double-stranded DNA molecule, and the other
    which binds to the other strand of the DNA
    molecule, all four nucleotides and a thermostable
    DNA polymerase.
  • The primers must be unique to the DNA being
    amplified and they flank the region of the DNA to
    be amplified.

48
PCR
  • The PCR reaction has three basic steps
  • Denature when you denature DNA, you separate it
    into single strands (SS).
  • In the PCR reaction, this is accomplished by
    heating at 950 C for 15 seconds to 1 minute.
  • The SS DNA generated will serve as templates for
    DNA synthesis.
  • Anneal to anneal is to come together through
    complementary base-pairing (hybridization).
  • During this stage in the PCR reaction the primers
    base-pair with their complementary sequences on
    the SS template DNA generated in the denaturation
    step of the reaction.

49
PCR
  • The primer concentration is in excess of the
    template concentration.
  • The excess primer concentration ensures that the
    chances of the primers base-pairing with their
    complementary sequences on the template DNA are
    higher than that of the complementary SS DNA
    templates base-pairing back together.
  • The annealing temperature used should ensure that
    annealing will occur only with DNA sequences that
    are completely complementary. WHY?
  • The annealing temperature depends upon the
    lengths and sequences of the primers. The longer
    the primers and the more Gs and Cs in the
    sequence, the higher the annealing temperature.
    WHY?
  • The annealing time is usually 15 seconds to 1
    minute.

50
PCR
  • Extension during this stage of the PCR
    reaction, the DNA polymerase will use dNTPs to
    synthesize DNA complementary to the template DNA.
  • To do this DNA polymerase extends the primers
    that annealed in the annealing step of the
    reaction.
  • The temperature used is 720 C since this is the
    optimum reaction temperature for the thermostable
    polymerase that is used in PCR. Why is a
    thermostable polymerase used?
  • The extension time is usually 15 seconds to 1
    minute.
  • The combination of denaturation, annealing, and
    extension constitute 1 cycle in a PCR reaction.

51
PCR
  • Most PCR reactions use 25 to 30 of these cycles
    to amplify the target DNA up to a million times
    the starting concentration.

52
PCR
53
PCR
54
Molecular Biology
  • DNA sequencing used to determine the actual DNA
    sequence of an organism. Using a computer, one
    can predict protein sequences and functions based
    on the nucleic acid data.
  • The most commonly used sequencing method is the
    dideoxy method.
  • This method uses dideoxy nucleotide triphosphates
    (ddNTPs) which have an H on the 3 carbon of the
    ribose sugar instead of the normal OH found in
    deoxynucleotides (dNTPs).
  • Dideoxynucleotides are chain terminators.
  • In a synthesis reaction, if a dideoxynucleotide
    is added instead of the normal deoxynucleotide,
    the synthesis stops at that point because the
    3OH necessary for the addition of the next
    nucleotide is absent.

55
Deoxy versus dideoxy
56
DNA synthesis
57
DNA sequencing continued
  • In the dideoxy method of sequencing, the template
    DNA that is to be sequenced is mixed with a
    primer complementary to the template DNA and the
    four normal deoxynucleotides, one of which is
    radioactively labeled for subsequent
    visualization purposes.
  • This mixture is then splint into four different
    tubes that are labeled A, C, G, and T. Each tube
    is then spiked with a different
    dideoxynucleotide (ddATP for tube A, ddCTP for
    tube C, ddGTT for tube G, or ddTTP for tube T).
  • DNA polymerase is added and using the DNA
    template and its complementary primer, the
    synthesis of new strands of DNA complementary to
    the template begins.
  • Occasionally a dideoxynucleotide is added instead
    of the normal deoxynucleotide and synthesis of
    that strand is terminated at that point.

58
DNA sequencing continued
  • In the tube containing ddATP, some percentage of
    newly synthesized molecules will get a ddATP in
    each place that there is a T in the template DNA.
  • The result is a set of new DNA molecules in tube
    A, each of which ends in an A.
  • A similar type of reaction occurs in the three
    other tubes to result in molecules that end in C,
    G, and T in tubes C, G, and T respectively.
  • After the synthesis reactions are complete, the
    products of the four different tubes are loaded
    onto four adjacent lane of a polyacrylamide gel
    and the different fragments are separated by
    size.
  • The sequencing gel is able to resolve fragments
    that differ in size from each other by only one
    base.

59
DNA sequencing continued
  • After electrophoresis to separate the fragments
    by size, the fragments are visualized by exposing
    the gel to photographic film (Remember that one
    nucleotide was radioactively labeled).
  • All fragments in lane A will end in an A,
    fragments in lane C will all end in a C,
    fragments in lane G will all end in a G, and
    fragments in lane T will all end in a T.
  • The sequence of the DNA is read from the gel by
    starting at the bottom and reading upward.

60
Dideoxy DNA Sequencing
61
DNA sequencing
62
DNA sequencing
  • Automated DNA sequencing in automated DNA
    sequencing a radioactive deoxynucleotide is not
    used and all four dideoxy reactions are done in a
    single tube.
  • This is possible because each dideoxynucleotide
    is labeled with a different flourescent dye.
  • Therefore the dye present in each synthesized
    fragment corresponds to the dye attached to the
    dideoxynucleotide that was added to terminate the
    synthesis of that particular fragment.
  • The contents of the single tube reaction are
    loaded onto a single lane of a gel (or capillary)
    and electrophoresis is done.
  • A flourimeter and computer are hooked up to the
    gel (or capillary) and they detect and record the
    dye attached to the fragments as they come off
    the gel.
  • The sequence is determined by the order of the
    dyes coming off the gel.

63
Automated DNA sequencing
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