LECTURE 4:
Methods used in Virology
Viro100:
Virology
3 Credit hours
NUST Centre of Virology & Immunology
3. Centrifugation
• After a virus has been propagated it is usually
necessary to remove host cell debris and
other contaminants before the virus particles
can be used for laboratory studies, for
incorporation into a vaccine, or for some other
purpose.
• Many virus purification procedures involve
centrifugation; partial purification can be
achieved by differential centrifugation and a
higher degree of purity can be achieved by
some form of density gradient centrifugation
Partial Purification by Differential Centrifugation
Purification of Virion by Density Gradient
Centrifugation
Rate zonal centrifugation involves layering the preparation on top of a pre-formed
gradient. Equilibrium centrifugation can often be done starting with a suspension of
the impure virus in a solution of the gradient material; the gradient is formed during
centrifugation.
4. Structural investigation of cells and
virion
• Light Microscopy
• Electron Microscopy
• Transmission Electron Microscopy
• Scanning Electron Microscopy
• X-ray crystallography
• Nuclear Magnetic Resonance
• Atomic Force Microscopy
Light Microscopy
• Virions are beyond the limits of resolution of light
microscopes
• Light microscopy has useful applications in
detecting virus infected cells
• Cytopathic effects
• Confocal microscopy
• The principle of this technique is the use of a
pinhole to exclude light from out of focus regions
of the specimen.
• Laser
Electron Microscope
• Structure of virions or of virus infected cells
involve electron microscopy
• Negative staining is carried out by using heavy
metal containing compounds
• Potassium phosphotungstate and ammonium
molybdate
• It generate contrast
• Negative staining techniques have generated
many high quality electron micrographs
X-ray Crystallography
• Detailed information about the three dimensional
structures of virions (and DNA, proteins and DNA
– protein complexes)
• Crystal of the virions or molecules
• crystal is placed in a beam of X-rays, which are
diffracted by repeating arrangements of
molecules/atoms in the crystal
• Analysis of the diffraction pattern allows the
relative positions of these molecules/atoms to be
determined
The diffraction information is fed into a computer, where 3-d
coordinates can then be calculated based on electron densities that
tend to diffract the x-rays more than anything else. The analysis
integrates all of this information to yield a prediction of the
complete 3-d structure
5. Electrophoretic techniques
• to separate proteins by charge and or size
• to separate a mixed population of DNA and
RNA fragments by length, to estimate the size
of DNA and RNA fragments
• DNA Gel electrophoresis
• SDS PAGE
• Sodium dodecyl sulphate, Polyacrylamide gel
electrophoresis
SDS PAGE
DNA Gel Electrophoresis
Thank You!