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Methods for Studying Viruses

Living Host System

Viruses require living host system due to:
 Acellular nature
 Lack of metabolic machinery
 Only genome in its structure

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Virus Cultivation
Cell Culture

Eggs

Animals

Unculturable

Making a Monolayer

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(a) Procedure for Inoculating Cell Culture in Petri Dish

(b) Plaques on Culture Dish

Indicators of Virus Infection

Cytopathic Effect (CPE)

Synthesis of viral proteins

Hemadsorption

Inclusion Bodies

Cell Transformation

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Cytopathic Effect

Cytopathic Effect

CELL ROUNDING HEMADSORPTION

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Intracytoplasmic & Nuclear

Inclusion Bodies

CELL TRANSFORMATION

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Routes of Inoculation of
Embryonated Egg

Inoculating Embryonated Egg

Membrane

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Normal & Viral Infected

Chorioallantoic Chick Membrane

Purification & Enumeration of

Viruses
Properties of viruses that lend themselves to isolation
techniques
 Size
 Often more resistant to chemicals, denaturing
treatments, and enzymes (nucleases and proteases)
 Coated with surface proteins

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Isolation Techniques
Differential centrifugation (acc. to size)
Density gradient centrifugation (acc. to size and
buoyant density)
Rate zonal gradient centrifugation (acc. to
sedimentation rates and density)
Precipitation (ammonium sulfate, polyethylene glycol)
Denaturation/removal of contaminants (chloroform,
Denaturation/removal
butanol,….
butanol ,…. to eliminate proteins and lipids)
Enzymatic digestion of cell constituents (nucleases,
proteases)

Ultrastructural Studies
1. Physical methods
2. Chemical methods
3. Electron microscopy

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Differential Centrifugation

Biochemical - Density Gradient

Separation

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Virus Detection and

Quantification:
Physical Methods
Electron Microscopy – High concentrations,
cumbersome, slow
Hemagglutination – Fast, easy but not
generally applicable
Immunoassay – Fast, easy, reliable and
widely applicable
PCR – Fast, extremely sensitive.
Can be quantitative

Electron Microscopy
Developed in the 1930s, overcome the fundamental limitation of
light microscopes, i.e. inability to resolve individual virus particles
owing to physical constraints caused by the wavelength of visible
light illumination & the optics of the instruments.
The first electron micrograph of a virus (TMV) was published in
1939.
Magnifications of over 100,000 times.
Two fundamental types
 Transmission electron microscope (TEM)
(TEM)
 Scanning electron microscope (SEM).

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Electron
Microscopy

Hemagglutination

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Hemagglutination

IMMUNOFLUORESENCE

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The Polymerase Chain Reaction

(PCR)

Denaturation of TMV

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Virus Detection & Quantification

Biological Methods
Plaque Assay

TCID50

LD50

Plaque Assay
One virion infects one
cell.
Progeny infect
surrounding cells.
After several cycles,
enough cells have been
killed to produce a
“plaque” visible to the
naked eye.

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Plaque Assays

Cell/virus –titrate infectious units

Uninfected Infected

Plaque Assay

Plaque assays are usually over layered with

agarose

Syncytia Assay

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B. Assays and detection

1. Plaque assay
a. End point dilution
b. Plaque forming units (PFU)

2. Agglutination

3. Infectivity eg: LD50

Syncytia
4. Cytopathic effect (CPE)

TCID50 Assay
Variation of the Plaque Assay

Method:
 Several dilutions of virus are prepared.
 Several culture tubes are inoculated with each dilution of
virus.
 After allowing time for virus growth, the number of tubes
showing virus growth is determined.
 Virus titer is the reciprocal of the dilution where 50% of
the tubes are infected.

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Titration: TCID50 Assay

1 8/8 = 100%

10 8/8 = 100%

103 8/8 = 100%

104 8/8 = 100%

105 8/8 = 100%

106 8/8 = 100%

107 6/8 = 63% TCID50

108 2/8 = 25%

109 0/10 = 0%

1010 0/10 = 0%

1011 0/10 = 0%

1012 0/10 = 0%

Host/virus interactions – LD50

Metabolic Labeling
Proteins: Radioactive amino acids,
[35S]Methionine
[35S] Methionine
Nucleic Acids: Radioactive
nucleotides, [32P]ATP

Immunological
• ELISA
• Western Blot
• Antibody Neutralization

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Molecular Assays
Is the genetic material isolated from the virus infectious?
Assay for viral genome
 RNA verses DNA

 Northern blot & RT/PCR, Southern blot and PCR

Sequence analysis
 Mutation analysis

Molecular Assays – Quantitative PCR

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Serological/Immunological Methods
Subsequently, many improved detection methods for viruses
were developed, for example:
Complement fixation tests
Radioimmunoassays
Immunofluorescence (direct detection of virus antigens in
infected cells or tissue)
Enzyme--linked immunosorbent assays (ELISAs)
Enzyme
Radioimmune precipitation
Western blot assays

Serological Methods

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Serological Methods

Serological Methods

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Serological Methods

Monoclonal Antibodies

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Hybridization Techniques
Nucleic acid-
acid-centred technology offers significant advances
in detection of viruses & virus infections involving nucleic acid
hybridization techniques.
There are many variants of this basic idea, but essentially, a
hybridization probe, labelled in some fashion to facilitate
detection, is allowed to react with a crude mixture of nucleic
acids.
The specific interaction of the probe sequence with
complementary virus-
virus-encoded sequences, to which it binds
by hydrogen-
hydrogen-bond formation between the complementary
base pairs, reveals the presence of the virus genetic
material.

Hybridization Techniques

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Bioinformatics
Because of the repetitive, digitized nature of nucleotide
sequences, computers are the ideal means of storing &
processing this information.
'Bioinformatics' is a broad term coined in the 1980's to
describe any application of computers to biology - anything
from artificial intelligence & robotics to genome analysis.
More specifically, the term applies to computer manipulation
of biological sequence data, including protein structural
analysis.
Bioinformatics permits the inference of function from the
linear sequence, & is thus central to all areas of modern
biology.

Bioinformatics
Computers are used increasingly to make predictions
based on nucleotide sequences, including:
 detecting the presence of open reading frames
 the amino acid sequences of the proteins
 control regions of genes such as promoters & splice
signals
 the secondary structure of proteins & nucleic acids

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Three--dimensional structure of the DNA binding domain of

Three
SV40 T-
T-antigen:

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