VIRAL

PROPAGATION
Viruses are extremely small particles.

Requires a high concentration (1011 virions/ml) +

electron microscope.

Tedious method to directly visualize.

Indirectly methods are preferred:

 Nucleic acids based testing

 Proteins based testing

 Lipids based testing

 BASIC TECHNIQUES TO IDENTIFY
VIRUSES

Three basic strategies:

A. Cell culture system

Animal cell culture

Organ Culture

Whole organisms

B. Serology

C. Nucleic acids
A. CELL CULTURE
SYSTEM
Virologists need to have sufficient viral titer to study various

aspects.

Also termed as viral propagation or viral horticulture.

To propagate viruses, host cells are mandatory.

Cell culture depends on type of virus under study.

Phages are supplied with bacterial cultures, plant viruses may be supplied

with specially cultivated plants or with cultures of protoplasts (plant cells

from which the cell wall has been removed), while animal viruses may be

supplied with whole organisms, such as mice, eggs containing chick

embryos or insect larvae. For the most part, however, animal viruses are

grown in cultured animal cells.

Cell culture system, in turns, comprises:

1. Cell culture

2. Whole organism

3. Organ culture
1. WHOLE ORGANISM CULTURE
Host-viral interaction---a complex phenomenon.

 Used to identify cellular check points.

 Modulated cellular signaling.

 Abrupt homeostasis.

To develop anti-viral drugs, inhibitors and vaccines.

 CONTD…

Organisms of interest:
 Mice, guinea pigs, chimpanzees and Monkey.

 Disadvantages:
 Very expensive
 lack of genetic diversity.
 Ethical issues.
2. Organ culture
ORGAN CULTURE

Organ cultures have the advantage of maintaining the differentiated

state of the target cell.

There are technical difficulties in their large scale use, and as a result
they have not been widely employed
3. CELL CULTURE

Cells to be cultured are grown in physiological conditions.

Temperature 37°C & 5% CO2.

Serum supplementation.

Mainly supplemented with Fetal bovine serum (FBS)---5-10%.

Some antibiotic like penicillin may also be employed.

Continuous cell lines

 Generally about 100 generations/cell culture.

 Needs to be preserved---cryopreservation.

 Huh7, Huh.8, HeLa, Ragiv, HepG2 cells, etc.

HOW CELLS ARE
CULTURED
See animation
B. Serology based
testing of viruses
Antibodies (Abs) are proteins
produced by the immune system
of higher vertebrates in response
to foreign materials (antigens) Plasma
which those cells encounter.

Leukocytes + Platelets

Each Antibody corresponds to a

particular region of an antigen
known as epitope. RBCs
 PRINCIPLE OF AB
TESTING
1. Neutralization test
 2. ELISA BASED TESTING
Panel of antibodies is being used
to identify an unknown virus
that is attached to the surface of
the plastic well. Here two
antibodies are used: the primary
antibody is specific for virus and
is unlabelled; the secondary
antibody is specific for
conserved epitopes and is
covalently linked to an enzyme.

https://www.youtube.com/watch?v=RRbuz3VQ100
This makes the ELISA more sensitive as several molecules of secondary
antibody (and associated enzyme) can bind to one molecule of primary
antibody. The colored product that results from reaction of the enzyme
with added substrate is proportional to the amount of primary antibody
bound and can be measured spectrophotometrically
C. Nucleic Acid based testing

https://www.youtube.com/watch?v=DkT6XH
Wne6E
WHY NUCLEIC ACID BASED
TESTING

While handling viruses, need to have the right procedure and

approach.

ELISA …quite rapid result.

 Frequency of false positive and false negative results is quite high.

 Not used for genome specificity.

 Cant be used to propagate gene of interest.

Nucleic acid based testing involved Polymerase chain reaction (PCR).

Straight forward mechanism.

Using the template gene, millions of copies are generated.

Used to detect and amplify targeted gene.

Genome differentiation is possible.