METHOD USED IN VIROLOGY • High Carbon dioxide (by a special

incubator)
VIRUS IDENTIFICATION

Cytopathic effects
- Microscopic or macroscopic degenerative
changes or abnormalities in host cells and tissues
Serological tests
- Detect antibodies against viruses in a patient.
- Use antibodies to identify viruses in
neutralization tests, viral hemagglutination, and
Western blot.
Nucleic acids KINDS OF CELL CULTURE
- RFLPs Cell Monolayer
- PCR • The cells are then suspended in culture
medium and placed in plastic flasks or
CYTOPATHIC EFFECT
covered plates. Attached to the plastic to
▪ Observe on a high power microscopy. form a monolayer.
▪ Changes in the cell • Commonly used cell lines double in
▪ Used to monitor the progress of infection. number in 24 to 48 h in such media
▪ Other viruses do not have the obvious • E.g Epithelial cell and Fibroblastic cells
cytopathic effect.
Primary culture
 Arenaviridae
 Paramyxoviridae • They have a limited life span, usually no
 Retroviridae more than 5 to 20 cell divisions.
• Examples are monkey kidneys, human
embryonic amnion and kidneys, human
foreskins and respiratory epithelium, and
chicken or mouse embryos.
• Used for vaccination preparation.
• Eg Poliovirus in monkey kidney cells
Continuous Cell line

• A single cell type that can be propagated

indefinitely in culture.
• E.g. HeLa Cells

VIRUS ISOLATION AND CULTURE

CELL CULTURE
▪ Used are from continuous cell lines derived
from humans and other animal species.
▪ Examples:
• HeLa Cells
• Taken from Henrietta Lacks, a woman
with cervical carcinoma.
▪ Important note for the cell medium
• Maintain osmotic pressure.
• pH
 EMBRYONATED EGGS
▪ At 5 to 14 days after fertilization, a hole is
drilled in the shell and virus is injected into
the site appropriate for its replication.
▪ Virus propagation for influenza virus
▪ For vaccine production

LABORATORY ANIMALS

▪ Use of monkeys in the study of poliomyelitis ▪ MODIFIED PLAQUE ASSAY

▪ Development of hepatitis B virus in  Fluorescent focus assay - a modification
chimpanzees. of the plaque Two hits assay, can be done
more rapidly and is useful in determining
ASSAY OF VIRUSES
the titers of viruses that do not form
TWO MAIN TYPES OF ASSAY FOR plaques.

DETECTING VIRUSES  Infectious center assay - used to

determine the fraction of cells in a culture
1. BIOLOGICAL that are infected with a virus.
 Plaque assay  Transformation assay - useful for
 End-point titration - do not measure non- determining the titers of some retroviruses
infectious particles. that do not form plaques.
 Viral Titer - the concentration of a virus ▪ END POINT DILUTION ASSAY
in a sample.  A measure of virus titer before the
2. PHYSICAL development of the plaque assay.
 Electron microscopy  The result of the assay can be expressed
 Immunological methods - measures in terms of 50% lethal dose (LD 50) per
both infectious and non-infectious milliliter or 50% paralytic dose (PD 50) per
particles milliliter, end points of death and paralysis,
 End point is the dilution of virus that
BIOLOGICAL affects 50% of the test units.

▪ PLAQUE ASSAY
- Modified by Renato Dulbecco (1952)
- To determine the titer of bacteriophage
stocks for use of animal virology

After When the

Monolayers Each
of cultured
removal of original
infectious
PHYSICAL
cells are the infected cells
particle
incubated inoculum, release new
produces
▪ ELECTRON MICROSCOPY
the cells are progeny
with a a circular - Provide detailed views of the smallest
preparation covered particles, the
with nutrient zone of bacteria, viruses, internal cellular
of virus to gel restricts
medium the spread of infected structures, and even molecules and large
allow
containing a viruses to cells, a atoms.
adsorption plaque.
to cells. supplement. neighboring - Ultrastructure
uninfected cell.
Two types:  Used in the Avian Influenza A (H7N9)
1. Transmission Electron Microscope in 2013.
2. Scanning Electron Microscope ▪ IMMUNOSTAINING
TRANSMISSION ELECTRON MICROSCOPE  Antibodies can be used to visualize
▪ Generates a viral proteins in infected cells or
beam of
electrons that tissues.
ultimately  Direct Immunostaining - an antibody
produces an
image on a that recognizes a viral protein is
fluorescent coupled directly to an indicator such as
screen.
▪ Dense Area: a fluorescent dye or an enzyme.
Block electrons (Dark Area)  Indirect Immunostaining - a second
▪ Less Dense: Screen fluoresces more
brightly. antibody is coupled to the indicator.
▪ Uses Ultramicrotome (there is sectioning) - Sensitive as compared to Direct
HEMMAGGLUTINATION Immunostaining.
▪ Members of the
Adenoviridae,
Orthomyxoviridae, and
Paramyxoviridae
▪ Viruses bind on the RBC
and can form lattice
▪ ENZYME IMMUNOASSAY
formation.  Detection of viral antigens or antiviral
▪ Example: Influenza virus - due to antibodies can be accomplished by
solid-phase methods, in which antiviral
glycoprotein called hemagglutinin. antibody or protein is adsorbed to a
SEROLOGICAL METHODS plastic surfac

▪ VIRUS NEUTRALIZATION
 Virus is inoculated to animal and
produce antibodies.
 Antibodies bind to virus particle and
can cause neutralization.
 End point: The highest dilution
DETECTION OF VIRAL NUCLEIC ACID
antibody inhibits the development of
POLYMERASE CHAIN REACTION
cytopathic effect in cells or virus. - Specific oligonucleotides are used to
▪ HEMMAGGLUTINATION INHIBITION amplify viral DNA sequences from infected
cells or clinical specimens.
 Antibodies against viral proteins with - Cycle
hemagglutination activity can block the ✓ Annealing

ability of virus to bind red blood cells. ✓ Extension

 It is the method of choice for assaying ✓ Thermal

denaturation
antibodies to any virus that causes
hemagglutination.
 DNA MICROARRAYS
▪ This approach provides a method for
studying the gene expression profile of a
cell in response to virus infection.
▪ In this method, millions of unique viral
DNA sequences fixed to glass or silicon
wafers are incubated with complementary
sequences amplified from clinical and
environmental samples.
▪ Detected by using fluorescent molecules
incorporated into amplified nucleic acids.
VIRUS PURIFICATION
➢ Four commonly used methods
1. Differential centrifugation and density
gradient centrifugation
2. Precipitation of viruses
3. Denaturation of contaminants
4. Enzymatic digestion of cell constituent
DIFFERENTIAL CENTRIFUGATION
- separates based on size.

DENSITY GRADIENT CENTRIFUGATION

- involves centrifuging particles (such as
virions) or molecules (such as nucleic
acids) in a solution of increasing
concentration, and therefore density.