Basic Techniques to Grow Viruses and Study VirusHost Interactions

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Basic Techniques to Grow Viruses and Study
Virus-Host Interactions
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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)

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Growth of Viruses, continued

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

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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.
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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.

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Making a primary cell line
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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.

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Making a continuous cell line
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Tissue Culture Cells
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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.

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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.

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Serial dilutions
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Animal Virus Plaque Assay
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Plaque assay results
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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.

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Hemagglutination assay
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Hemagglutination assay
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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.

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Hemagglutination inhibition assay
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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.

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Direct immunofluorescent antibody test
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Indirect immunofluorescent antibody test
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Immunofluorescence
?
?
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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.

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Direct (sandwich) ELISA
(virus?)
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Indirect ELISA
virus
against virus?
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Indirect versus direct (sandwich) ELISA
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ELISA (sandwich method to detect Ag)
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ELISA (indirect)
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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.

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Western immunoblot
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Western Blot
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Indirect Western immunoblot for HIV diagnosis
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Indirect Western immunoblot for HIV diagnosis
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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.

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Molecular Sieve Chromatography
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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.

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Centrifugation
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Equilibrium density gradient centrifugation
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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.

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Electrophoresis
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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.

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Affinity chromatography
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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

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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.

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In situ hybridization
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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.

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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.

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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.

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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.

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PCR

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

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PCR
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PCR
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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.

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Deoxy versus dideoxy
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DNA synthesis
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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.

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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.

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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.

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Dideoxy DNA Sequencing
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DNA sequencing
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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.

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Automated DNA sequencing