Laboratory Diagnosis Laboratory Diagnosis of Viral Infectionof Viral Infection
Laboratory Diagnosis Laboratory Diagnosis of Viral Infectionof Viral Infection
Detection – Isolation - SerologyDetection – 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 animals
NT
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 detectionFluorescent antibody test. ELISAImmunodiffusion.
• Nucleic acid
1- 5- PCR.
1. EM detection of corona virus
Hepatitis B virus
EM picture of rabies virus
2. Detection of virus byImmunofluorescent
Technique
Diagramatic presentation of IF technique
2. Detection of virus byImmunofluorescent
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
1 2 3
B
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
• Incubate for one hour then inoculate each into cell culture tubes, incubate and observe daily.
2 31
Principle of Neutralization test
Rabid Virus
Negi bodies
IF staining of rabies infected
brain cells
Diagramatic presentation of rabies
virus
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
HA
HAI
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
SerologyNeutralization
• 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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