Lecture 3

Laboratory
diagnosis of viral
infection
Saleh Bahaj
Prof. of Medical
Microbiology/Immunology
 Objectives
By the end of this lecture the students
will be able to:-
I) Direct demonstration of virus

II) Virus Isolation

III) Serological tests (Ab)
IV) Test yourself
 Laboratory diagnosis
of viral infection
I) Direct demonstration
of virus in clinical
specimen
I) Direct demonstration of virus
1) Detection of the virus particle
2) Detection of inclusion bodies
3) Detection of specific viral Ag
4) Detection of Nucleic acid
of the virus
Laboratory diagnosis
of viral infection
I) Direct demonstration
of virus
1) Detection of the virus particle
I) Direct demonstration of virus

1
Detection of the
virus particle
By E.M.
(Clinical specimen)
I) Direct demonstration of virus

2
Detection of the
virus particle
By Immunoelectron
microscopy
(Ab)
Laboratory diagnosis
of viral infection
I) Direct demonstration
of virus
2) Detection of inclusion bodies
I) Direct demonstration of virus

3
Detection
Inclusion bodies
By L.M.
Negri bodies
Laboratory diagnosis
of viral infection
I) Direct demonstration
of virus
3) Detection of specific viral Ag
I) Direct demonstration of virus

4
Detection of
Viral antigen
e.g. HBsAg
(ELISA)
Laboratory diagnosis
of viral infection
I) Direct demonstration
of virus
4) Detection of Nucleic acid
of the virus
I) Direct demonstration of virus

5a
Detection of
viral nucleic acid
(PCR)
I) Direct demonstration of virus

5b
Hybridization
reaction
(Tissue)
II) Virus Isolation
(Cultivation of the
viruses)
 II) Cultivation of the
viruses

Virus isolation
needs
living cells
 II) Cultivation of the
viruses
1) Laboratory animals
2) Embryonated eggs
3) Cell (tissue) culture
II) Virus Isolation
(Cultivation of the
viruses)
1) Laboratory animals
 1) Laboratory animals

Interference with immune system?

II) Virus Isolation
(Cultivation of the
viruses)
2) Embryonated eggs
2) Embryonated eggs

Fertilized egg
 2) Embryonated eggs
1)Chorioallantoic 1
membrane
2
2)Aminiotic cavity
3) Allantoic
inoculation 3
4) Yolk sac inoculation 4
 2) Embryonated eggs

No immune response
II) Virus Isolation
(Cultivation of the
viruses)
3) Cell (Tissue) culture
 3) Cell (Tissue) culture

It consists of
single layer
(monolayer cells)
3) Cell (Tissue) culture

Trypsin
Pieces
Growth media
low of contact
inhibition

Monolayer cell
Types of cell culture
A- Primary cell lines
 A- Primary cell lines

Obtained from
organ fragments
e.g. monkey
kidney
 A- Primary cell lines

Only divide for

several passages
(10) and then
degenerate
Types of cell culture
B- Human diploid cell lines
(2 cell lines)
nd
 B- Human diploid cell lines

Derived from
human embryo
tissues
(Fibroblast)
 B- Human diploid cell lines

Only divide about

50 -100 passages in
culture e.g. human
embryo lung 100
tissues.
Rapid growth & increase
subculture
Types of cell culture
C- Continuous cell lines
(3 cell lines)
rd
 C- Continuous cell lines

Hela cells
Henrietta Lacks
1951
(cervical carcinoma)
 C- Continuous cell lines

Hela cells
Divide
indefinitely
Detection of
viral growth
 Detection of viral growth

1- Cytopathogenic
effect (CPE)

Changes in cells
 1- Cytopathogenic effect (CPE)

a) Cell death
b) Rounding
c) Syncytium
d)Cell transformation
 1- Cytopathogenic effect (CPE)

a) Cell death

Detachment from
glass surface
e.g. poliovirus
 1- Cytopathogenic effect (CPE)

b- Rounding

e.g. Adenovirus
 1- Cytopathogenic effect (CPE)

c- Syncytium

e.g. Measles &

mumps
 1- Cytopathogenic effect (CPE)

d- Cell transformation

Tumor viruses
 Detection of
viral growth
2) Plaque formation
 2) Plaque formation
 Virus infects cells

 When using vital stain

(neutral red)

 unstained areas for

infected cells
 Detection of
viral growth
3) Inclusion bodies
 3) Inclusion bodies
They are the site of
virus assembly

Intracytoplasmic
 rabies (Negri
bodies).
 3) Inclusion bodies

Intra- Intra- Both

cytoplasmic nuclear
 Detection of
viral growth
4) Haemadsorption
 4) Haemadsorption

Some virus
have
RBCs Haemagglutination
haemagglutinin (O-)
4) Haemadsorption
 Detection of
viral growth
5) Fluorescent-
antibody staining
 5) Fluorescent- antibody staining

F
Y
Y

YF
F

Y
F
 Detection of
viral growth
6) Neutralization test
6) Neutralization test

CPE
Tissue culture
 6) Neutralization test

Y
Y

No CPE
Y

Y Tissue culture
 Detection of
viral growth
7) Detection of viral Ag
7) Detection of viral Ag
Detection of
viral growth
8) Interference
 8) Interference
Virus
producing Tissue culture CPE
CPE

Virus
Virus in
producing
No
tissue culture
CPE CPE
 8) Interference
Sorry we are closed
The replication of one
virus in a cell usually
inhibits the
multiplication of
another virus entering
subsequently.
 8) Interference

1- Destroy the STOP

receptors
(Prevent the
attachment)
 8) Interference

STOP
2- Prevent
mRNA
formation
 Laboratory diagnosis
of viral infection

III) Detection of specific

Antibodies
(Serological tests)
 III) Serological tests

Serum
Detection Ab
Antibodies
 III) Serological tests

IgM
Early
(Recent)
 III) Serological tests

IgG
Past
 III) Serological tests
 Detection of rising titre
of IgG by ELISA

2nd sample after

7 days
1/80 1/320
 Exercise 1
The following virus infect our body,
discuss with your team the methods
of diagnosis of this virus.
 Answer 1
Specimen
I) Direct demonstration of the virus
1) Detection of the virus particle
2) Detection of inclusion bodies
3) Detection of specific viral Ag
4) Detection of Nucleic acid
of the virus
 Answer 1

II) Virus isolation

Cell culture
 Answer 1

III) Serological tests

1)Detection of viral Ag
2)Detection of viral Ab
V) Test yourself
 Q1
Direct demonstration of viral infection
can be done by
Detection of specific viral Ag
Detection of nucleic acid by PCR
Detection of viral particles by EM
Monocyte
Detection of Antibodies
2-3 days
 Q2
Direct demonstration of viral nucleic acid
in the infected tissue can be done by
EM
Light microscopy
Hybridization technique
Monocyte
Monocyte
PCR
2-32-3
days
days
 Q3
Transformation of infected cell by
oncogenic virus means
Uncontrolled cell growth
Cell deattachment
Inclusion bodies
Monocyte
Plaque formation
2-3 days
 Q4
Hemadsorption produced by a virus in an
infected cell means
Morphological changes
Formation of inclusion bodies
Attachment of erythrocytes to the
infected
Monocyte cells
2-3 days
Plaque formation
 Q5
Virus isolation needs

Nucleic acid detection

Tissue culture
Embryonated egg
Monocyte
Laboratory animal
2-3 days
 Q6
Polymerase chain reaction (PCR) is used for
detection of

Viral inclusion bodies

Viral specific Ag
Viral nucleic acid
Monocyte
Viral specific Aby
2-3 days
 Q7
All the following tests are used for detection
viral antibodies EXCEPT

Complement fixation test

Immunofluorescence test
PCR
Monocyte
ELISA
2-3 days
 Q8
Hybridization technique which is used for
diagnosis of viral infection depends on the use

Antibodies
Enzyme labelled antibodies
Nucleic acid probe
Monocyte
Antigens
2-3 days
 Q9
The most commonly used diagnostic
technique in the virus laboratory:
Virus isolation in cell culture
Electron microscopy
PCR & RT-PCR
Monocyte
Serological tests
2-3 days
 Q10
All the following primary cell line of cell
culture
Produced from organ fragment
Subcutured up to 10 passages
Derived from tumor cells
Monocyte
Derived from embryo
2-3 days tissue
 Objectives
By the end of this lecture the students
will be able to:-
I) Direct demonstration of virus

II) Virus Isolation

III) Serological tests (Ab)
IV) Test yourself
Th
an
k yo
u