Laboratory

Laboratory Diagnosis
Diagnosis
of
of Viral
Viral Infection
Infection
Detection – Isolation - Serology
 What is a virus?

It is a segment of either RNA or

DNA protected by a protein coat
and in some families of viruses a host
derived envelope with attached viral
proteins
It lacks:
- Protein synthesizing machinery
- Energy producing system
- No mitochondria
- No stores of amino acids, nucleotides energy
rich molecules
It is a compulsory intra cellular parasite
 It depends on three main
principles:
Direct detection Isolation on: Serology using:
of:

Virus particles • Tissue culture IF

Viral antigen • Chick embryo HI

Viral nucleic acid • Laboratory NT

animals

Cytopathology ELISA
 I. Direct detection of virus
particle
Direct detection of virus:
• particle,
• viral antigen,
• or viral nucleic acid in clinical
specimens
 I. Direct detection of virus
particle
Direct detection could be done by one of the following:
•
Particle
• Electron microscopy.
–
Antigen detection
•
Fluorescent antibody test.
ELISA
Immunodiffusion.
Nucleic acid
•

1- 5- PCR.
1. EM detection of
corona virus
Hepatitis B virus
EM picture of rabies
virus
 2. Detection of virus by
Immunofluorescent Technique

Diagramatic presentation of
IF technique
 2. Detection of virus by
Immunofluorescent Technique
IF staining of rabies
infected brain cells
3. ElISA
4. Immune diffusion
 5. Nucleic acid
techniques
• PCR

• Probe Hybridization
 II- Isolation and
identification
II- Isolation and identification of the virus
from clinical specimens: three main
systems are used for viral isolation:
1- Tissue culture.

2- Chick embryo.

3- Laboratory animals
 Tissue culture preparation:

From the desired tissue the following steps are followed:

• Mince into 1mm fragments.

• Incubate with proteolytic enzyme (trypsin) to disperse

the cells.

• Add growth media to make a cell suspension.

• Incubate in stationary flasks or tubes, cells settle on

the dependent surface and grow into confluent
monolayer.

• Re-disperse monolayer cells and increase number of

cultures for cell culture passage.
Virus isolation in tissue
culture cell line
 Viral identification:

This is achieved by:

(a) The effect on cell culture:
i.e. cytopathic effect,

(b) Neutralization test. This is based on the

neutralization of the virus infectivity by
mixing it with specific antibody before
inoculation into cultures.
Cytopathic effect
 Neutralization Test
Following virus isolation: 1.Divide culture yield into small volume in a set of
test tubes
2. Prepare the panel of antisera against which the virus isolate is to be
challenged
2. To each test tube add one antisera and leave one as a virus control and
one as serum control
1 2 3

B
• Incubate for one hour then inoculate
each into cell culture tubes, incubate
and observe daily.
1 2 3

Principle of Neutralization test

Rabid Virus

Diagramatic presentation
of rabies virus

IF staining of rabies
infected brain cells

Negi bodies
Isolation in embryonated
hen’s eggs
 Inoculation into the
amniotic cavity of the chick
embryo.
Inoculation into the yolk sac
of the chick embryo
A VIRUS INOCULATION BEING DROPPED
ONTO THE CHORIOALLANTOIC
MEMBRANE OF THIRTEEN DAY OLD
CHICK EMBRYO .
Herpes virus lesion on the
chorioallantoic membrane
 Haemagglutination
& Haemagglutination inhibition

HAI

HA
 III- Serological demonstration
of the antibodies by:

1- Immunofluorescence (IIF).
2- Enzyme immunosorbant assay (ELISA).
3- Haemagglutination inhibition test (HI).
4- Neutralization test (NT).
5- Complement fixation
 Serology
Neutralization
• Neutralization.

Standardized antiserum is used

 HI
• HI tests to detect specific
antibodies to a virus in the patient’s
serum
Main material used
• Standardized virus.
THANK YOU