PRACTICAL VIROLOGY
Introduction
Lab. 1
Laboratory Safety
• It is important to remember that whenever one
works with an infectious agent there is the
possibility of infection to oneself or to others by
negligence
• Furthermore, organisms that are referred to as
"nonpathogenic“ are still potential pathogens
• For example, certain "non-human“ viruses, such as
Newcastle disease virus (NDV), have been known to
cause conjunctivitis when inadvertently introduced into
the human eye
Laboratory Safety
• Cell cultures can also be potentially hazardous
since they can infect the laboratory worker with
an endogenous or a latent virus
• One should adopt the same precautions when
working with cell culture as when working with a
virus
General Laboratory Safety Procedures
• There must be NO EATING, DRINKING, SMOKING, OR
APPLYING OF COSMETICS IN THE LABORATORY.
• Unauthorized persons, particularly children and infants,
should NOT be allowed in the laboratory.
• NO MOUTH-PIPETING should be permitted due to the
Possibility of accidental ingestion of virus-contaminated
materials, cell-contaminated products, or toxic chemicals.
Automatic pipetors or pipeting bulbs must always be
used.
• Hands should be washed when coming in the laboratory,
after handling cells or virus, and before leaving the
laboratory.
General Laboratory Safety Procedures
• One should NOT walk around touching door
knobs and surfaces with contaminated gloves.
Contaminated gloves should be discarded in the
decontamination pan.
• Avoid touching the eyes, nose, mouth, or face
while working in the laboratory.
• Laboratory coats should be worn when doing cell
culture or virus Work. Laboratory coats should
NOT be worn while eating or outside of the
laboratory.
General Laboratory Safety Procedures
• Whenever working with syringes and needles,
special containers (that are sealed and
autoclavable) should be provided for their
disposal.
• DONOT replace the cap on the needle (this is
where many accidental injections occur). Drop
the uncapped syringe into the disposal container
immediately after use. These containers are then
autoclaved prior to disposal.
• Viruses, cells, or their products, should NEVER
be disposed of in the drainage system.
STERILIZATION
Autoclaving
• Autoclaving is used when ever possible,
especially when dealing with liquid
solutions and materials that should not
dry out.
• The use of Autoclave tape on all
materials to be autoclaved permits the
identification of those materials that
have been autoclaved and prevents the
accidental opening of contaminated
materials.
• The standard cycle is 121°C, 15 p.s.i.,
for 15 minutes.
Dry Heat Sterilization
• Effective for glassware
provided there are no
rubber or plastic-lined parts.
• The standard cycle is at
300°C for 2 hours.
• Proper loading of the oven
is also very important so as
to reduce insulating effects
Filtration
• Filtration (using a 0.22
μm membrane filter) is
used for aqueous
solutions and medium
components that are
heat labile.
ASEPTIC TECHNIQUE
• Even when sterile materials are available, proper
aseptic technique must be rigorously enforced in
order to avoid accidental contamination of cell
cultures
• Biohazard hoods are preferable for cell culture
work
• It is important to disinfect the working area with
an appropriate disinfectant just prior to and after
working.
• It is helpful to keep flasks and bottles open for as
short period as possible.
• Remember to flame the lips of the bottles, flasks,
and tubes after opening, and to use only sterile
pipets tips.
• If a sterile pipet tip accidentally touches a nonsterile surface, it should be discarded and
another one used in its place
VIRUSES
Viruses
• Virus is a small infectious
agent that replicates only
inside the living cells of
other organisms
• Viruses can infect all
types of life forms from
animals and plants to
bacteria
Viruses
• They are small, nanometer “nm” is the measuring unit
Reasons for Studying Viruses
• Viruses can infect all
cellular life forms
• It is undoubtedly the
case that the viruses
that have been
discovered represent
only a tiny fraction of
the viruses on the
Earth
(Virology Principles & Applications Book
Reasons for Studying Viruses
• Viruses are important agents of many human diseases
• Common cold
• Cancer, acute hepatitis, AIDS
• There is therefore a requirement to understand:
• the nature of viruses,
• how they replicate
• and how they cause disease
• This knowledge permits the development of effective
means for:
• Prevention, through the production of vaccines,
• Diagnosis, through the production of diagnostic reagents and
techniques
• and treatment of virus diseases, through the production of antiviral
drugs
Reasons for studying viruses
• Some viruses are studied because they have
useful current or potential applications
•
Phage typing of bacteria:
• Some groups of bacteria,
such as some Salmonella
• species, are classified into
strains on the basis of the
spectrum of phages to
which they are susceptible
Reasons for studying viruses
• Sources of enzymes:
• A number of enzymes used in
molecular biology are virus
enzymes (eg. reverse
transcriptases from retroviruses
and RNA polymerases from
phages)
• Pesticides:
• Some insect pests are controlled
with baculoviruses and myxoma
virus
Reasons for studying viruses
• Gene vectors for protein production:
• Viruses such as certain baculoviruses and
adenoviruses are used as vectors to take genes into
animal cells growing in culture
• Gene vectors for treatment of genetic diseases:
• Children with severe combined immunodeficiency have
been successfully treated using retroviruses as vectors
to introduce into their stem cells a non-mutated copy of
the mutated gene responsible for the disease
Reasons for studying viruses
• Anti-cancer agents:
• Genetically modified strains of viruses, such as herpes
simplex virus and vaccinia virus, are being investigated
for treatment of cancers
Study of Viruses
• Characteristics:
• Acellular
• Obligate intracellular parasites
• No ribosomes or means of protein synthesis
• No ATP generating system
• NOT ALIVE!
METHODS USED IN VIROLOGY
Cultivation of viruses
• Three methods:
• Living animals
• Chicken embryos
• Cell culture
Isolation of viruses
• Many viruses can be isolated as a result of their
ability to form discrete visible zones, plaques
(areas where cells are killed or altered by the
virus infection) in the host cells
Plaques formed by a phage in a
bacterial lawn
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
Partial purification of virions by differential centrifugation
Density gradient centrifugation
• Involves centrifuging particles (such as virions) in
a solution of increasing concentration, and
therefore density
Purification of virions by density gradient centrifugation
Structural investigations of cells and virions
• Light microscopy
• has useful applications in detecting
virus-infected cells, for example by
observing cytopathic effects
• Fluorescence microscopy
• Or by detecting a fluorescent dye linked
to antibody molecules that have bound
to a virus antigen
Electron microscopy
• Electron microscopy
• Many investigations of the structure of
virions or of virus-infected cells involve
electron microscopy
Electrophoretic Techniques
• Mixtures of proteins or nucleic acids can be
separated by electrophoresis in a gel composed
of agarose or polyacrylamide