Transcript Laboratory Diagnosis of Viral Infection

Laboratory Diagnosis
of Viral 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
of:
Isolation on:
Serology using:
Virus particles
 Tissue culture
IF
Viral antigen
 Chick embryo
HI
Viral nucleic acid  Laboratory
NT
Cytopathology
ELISA
animals
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. 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
Principle of Neutralization test
3
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:
12345-
Immunofluorescence (IIF).
Enzyme immunosorbant assay
(ELISA).
Haemagglutination inhibition test
(HI).
Neutralization test (NT).
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.
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