Veterinary Virology

Aris F. Miclat
Methods used in Virology
Cultivation of viruses
⚫ Animal cell culture
⚫ well developed
⚫ cells used are from continuous cell lines
derived from humans and other animal
species.
⚫ continuous cell lines consist of cells that have
been immortalized, either in the laboratory or
in the body.
Isolation of viruses
⚫ Many viruses can be isolated as a result of their ability
to form discrete visible zones (plaques) in layers of
host cells.
⚫ It is generally assumed that a plaque is the result of the
infection of a cell by a single virion.
⚫ All virus produced from virus in the plaque should be a
clone, in other words it should be genetically identical.
This clone can be referred to as an isolate, and if it is
distinct from all other isolates it can be referred to as a
strain.
⚫ Plaques can be formed by many animal viruses in
monolayers if the cells are overlaid with agarose
gel to maintain the progeny virus in a discrete
zone.

⚫ Plaques can also be formed by phages in lawns of

bacterial growth.
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.
Differential centrifugation
⚫ involves alternating cycles of low-speed
centrifugation, after which most of the
virus is still in the supernatant, and
high-speed centrifugation, after which
the virus is in the pellet.
Density gradient centrifugation
⚫ Density gradient centrifugation involves
centrifuging particles (such as virions) or
molecules (such as nucleic acids) in a solution of
increasing concentration, and therefore density.
⚫ The solutes used have high solubility: sucrose is
commonly used.
⚫ There are two major categories of density
gradient centrifugation: rate zonal and
equilibrium (isopycnic) centrifugation.
Structural investigations
of cells and virions
⚫ Light microscopy
⚫ useful applications in detecting virus-infected cells, for
example by observing cytopathic effects
⚫ Confocal microscopy
⚫ makes use of a pinhole to exclude light from out of-focus
regions of the specimen.
⚫ most confocal microscopes scan the specimen with a laser,
producing exceptionally clear images of thick specimens and
of fluorescing specimens.
⚫ The techniques can be used with live cells and can be applied
to investigations of protein trafficking, with the virus or cell
protein under investigation carrying a suitable label, e.g.
green fluorescent protein (a jellyfish protein).
⚫ Electron microscopy
⚫ large magnifications
⚫ negative staining techniques generate contrast by using
heavy-metal-containing compounds, such as potassium
phosphotungstate and ammonium molybdate

⚫ X-ray crystallography
⚫ reveals detailed information about the three-dimensional
structures of virions (and DNA, proteins and DNA–protein
complexes).
⚫ This technique requires the production of a crystal of the
virions or molecules under study.
⚫ The crystal is placed in a beam of X-rays, which are
diffracted by repeating arrangements of molecules/atoms in
the crystal.
Electrophoretic techniques
⚫ Mixtures of proteins or nucleic acids can be separated by
electrophoresis in a gel composed of agarose or
polyacrylamide. In most electrophoretic techniques each
protein or nucleic acid forms a band in the gel.
⚫ The molecular weights of the protein or nucleic acid
molecules can be estimated by comparing the positions of
the bands with positions of bands formed by molecules of
known molecular weight electrophoresed in the same gel.
⚫ The technique for estimating molecular weights of proteins
is polyacrylamide gel electrophoresis in the presence of the
detergent sodium dodecyl sulphate (SDS-PAGE).
Detection of viruses and virus
components
⚫ Detection of virions
⚫ Specimens can be negatively stained and examined in an
electron microscope for the presence of virions.
⚫ Limitations to this approach are the high costs of the equipment
and limited sensitivity.

⚫ Detection of infectivity using cell cultures

⚫ Infectivity is used to denote the capacity of a virus to replicate.
⚫ After incubation of an inoculated cell culture at an appropriate
temperature it can be examined by light microscopy for
characteristic changes in the appearance of the cells resulting from
virus-induced damage.
⚫ A change of this type is known as a cytopathic effect (CPE).
⚫ Detection of virus antigens
⚫ Virus antigens can be detected using virus-specific antisera or
monoclonal antibodies.
⚫ Antibodies can have many types of label attached and the
labels can be detected using a range of methods.

⚫ Detection of virus nucleic acids

⚫ Hybridization
⚫ Virus genomes or virus messenger RNAs (mRNAs) may be
detected using sequence-specific DNA probes carrying
appropriate labels.
⚫ Hybridization may take place on the surface of a membrane after
Southern blotting (DNA) or northern blotting (RNA).
⚫ Thin sections of tissue may be probed for the presence of specific
nucleic acids, in which case the technique is known as in situ
hybridization.
⚫ Polymerase chain reaction (PCR)
⚫ When a sample is likely to contain a low number of
copies of a virus nucleic acid the probability of detection
can be increased by amplifying virus DNA using a PCR.
Thank you!