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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