Depend on three principle techniques

Depend on three principle techniques

Direct demonstration of viral Ag or nucleic acid

Isolation and identification of the virus.

Serological demonstration of Ab to the virus.

• EM• Florescent Ab.• Double I.D.• Probing• PCR

• EM• Florescent Ab.• Double I.D.• Probing• PCR

• T.C.• Chick embryo• Lab. animal

• T.C.• Chick embryo• Lab. animal

• I.F.• ELISA• NT• HAI

• I.F.• ELISA• NT• HAI

SERUMCLINICAL SPECIMEN

A-Direct detection

1. Electron microscopy:

It show the detail structure of the virus.

Viruses diameter 10-200 nm can be resolved

A-Direct detection

1. Electron microscopy:

It show the detail structure of the virus.

Viruses diameter 10-200 nm can be resolved

Fig.1 Electron microscope

EM.picture of viruses

2) Fluorescent-antibody test

•Viral antigen in smears detected by direct immunofluorescent

technique (DIF).

•Specific fluorescencet stain with standard antiviral serum

• Examined by UV microscope.

Immunofluorescent technique

3) Double immunodiffusion

•V. antigen can be detected by precipitation against antiviral serum in agar gel

•Rapid method and takes 2-6 hours

4-ELISA Double antibody sandwitch technique for detecting antigen.

4-ELISA Double antibody sandwitch technique for detecting antigen.

5-Nucleic acid probe:DNA probes are pieces of nucleic acid labeled in some fashion that can find to structures of DNA that have complementary sequences. So unknown organism can be identified.

5-Nucleic acid probe:

DNA probes are pieces of nucleic acid labeled in some fashion that

can find to structures of DNA that have complementary sequences.

So unknown organism can be identified.

5-Nucleic acid probe:

DNA probes are pieces of nucleic acid labeled in some fashion that

can find to structures of DNA that have complementary sequences.

So unknown organism can be identified.

6-Polymerase chain reaction (PCR)

• It is a process of amplification of specific DNA sequence.

• The target could be amplified more than million fold.

• Quantitative PCR (Real time PCR) available to detect

viral load ( no. of viral particles/ml blood.)

Important in monitoring the progression of disease

and response to treatment.

6-Polymerase chain reaction (PCR)

• It is a process of amplification of specific DNA sequence.

• The target could be amplified more than million fold.

• Quantitative PCR (Real time PCR) available to detect

viral load ( no. of viral particles/ml blood.)

Important in monitoring the progression of disease

and response to treatment.

Cytological examination:

•Demonstration of characteristic cytological changes from smears of infected tissues.

•Inclusion bodies ( IB ),multinucleated( syncetial ) giant cells are the main changes.

• IB are masses of viral particles seen in nucleus or cytoplasm; intranuclear or intracytoplasmic,either acidophilic or basophilic in nature.

•Diseases as HSV, VZ virus and rabies produce cytologiccal changes .

B-Virus isolation

Specimen:

Body secretions or excretions, or from the site of lesion.

Taken on a swab to preserve the virus on transport media

(TC. Media)Specimen reach the lab as fast as possible.Specimen must be kept cold.Antibiotics and antifungal are added to the specimen in the

lab, avoid bact. contamination.Specimen is centrifuged to deposit the bact.

B-Virus isolation

Specimen:

Body secretions or excretions, or from the site of lesion.

Taken on a swab to preserve the virus on transport media

(TC. Media)Specimen reach the lab as fast as possible.Specimen must be kept cold.Antibiotics and antifungal are added to the specimen in the

lab, avoid bact. contamination.Specimen is centrifuged to deposit the bact.

Laboratory animals

Three main systems are used for virus isolation

Tissue culture Chick embryo

1-Tissue culture:

• TC- Cells from man or animal are grown as a single layer

(mono-layer) on the wall of the tubes or on one side of flat bottle.

Cells are incubated at 37C.

•Suspended in tissue culture media

1-Tissue culture:

• TC- Cells from man or animal are grown as a single layer

(mono-layer) on the wall of the tubes or on one side of flat bottle.

Cells are incubated at 37C.

•Suspended in tissue culture media

Virus TC-cell line incubated signs of growth identification and typing of virus.

Three main types of tissue culture cell lines:

1-Primary culture:

Cells are dispersed with trypsin.

Little cell division during growth.

It form mono layer on the surface of the glass tube.

After 2-3 weeks the cells degenerate and are discarded.

Three main types of tissue culture cell lines:

1-Primary culture:

Cells are dispersed with trypsin.

Little cell division during growth.

It form mono layer on the surface of the glass tube.

After 2-3 weeks the cells degenerate and are discarded.

2-Semicontinuous cell lines

Cells are usually fibroblasts derived from embryonic tissue

e.g human diploid cell line .

2-Semicontinuous cell lines

Cells are usually fibroblasts derived from embryonic tissue

e.g human diploid cell line .

3-Continuous cell line

Usually derived from malignant cellse.g Hela cells cervical

cancer.

Normal tissue culture cell line :

Virus growth is recognized by:

1) Cytopathic effect: CPE i.e. cell degeneration or death which

is recognized by

Rounding, shrinkage, ballooning

As the cell die, it is detached from the glass

Multi nucleated giant cell or syncetia.

Virus growth is recognized by:

1) Cytopathic effect: CPE i.e. cell degeneration or death which

is recognized by

Rounding, shrinkage, ballooning

As the cell die, it is detached from the glass

Multi nucleated giant cell or syncetia.

2) Haemadsorption

Seen with haemagglutinating viruses which mature at the cell surface.

Virus infected cell + Erythrocytes eryth. Adherent to the infected cell.

Virus infected cell

+

3) Haemagglutination

Haemagglutination test is done on the cell fluid to detect the

presence of haemagglutinating virus.

4)Interference

3) Haemagglutination

Haemagglutination test is done on the cell fluid to detect the

presence of haemagglutinating virus.

4)Interference

Haemagglutination pattern.

5) Immunofluorescence

Vs antigen is detected in infected cell by DIF.

6) Detection of virus specific NA: by PCR.

5) Immunofluorescence

Vs antigen is detected in infected cell by DIF.

6) Detection of virus specific NA: by PCR.

2. Chick embryo

Fertile hen`s egg

• Used before the advent of tissue culture.

• Susceptible to fewer no. of viruses.

2. Chick embryo

Fertile hen`s egg

• Used before the advent of tissue culture.

• Susceptible to fewer no. of viruses.

C- Serological Diagnosis:

Demonstration of virus antibody in patient serum.

A diagnosis of recent infection depend on:

1-Detection of IgM.2-Rising titre: Increase in level of antibody titre; at least four –fold rise.3-High stationary titre of antibody.

Serological tests used in serology:

1-IF (IIF).2-ELISA: Indirect ELISA.3-Haemagglutination-inhibition test.4-Neutralization test.

The ELISA test in a microplate.Microtitre plates of special quality and with flat bottomed wells are used.

The colour produced is the basis for interpretation of the reaction, which is read by a photometer. The four rows on the left are the controls.

The ELISA test in a microplate.Microtitre plates of special quality and with flat bottomed wells are used.

The colour produced is the basis for interpretation of the reaction, which is read by a photometer. The four rows on the left are the controls.