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 animals NT Cytopathology ELISA

I. 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.

Principle of Neutralization test 1 2 3 Principle of Neutralization test

Diagramatic presentation of rabies virus 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 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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