Lab Diagnosis of Viral Infection

Direct detection of virus/ viral Ag Serology

Cytopathology

Virus isolation of viral Ab & Identification of viruses

IF Tissue culture Chick embryo Lab animals HIT NT

EM Double Immunodiffusion ELISA ELISA NA probe PCR

Immunoblotting Techniques
For components of the antibody response Western blot (for detection of HIV antibodies) RIBA (for detection of HCV antibodies)

LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

Laboratory virological diagnosis depends on three principle techniques: 1.Direct demonstration of virus, viral antigens or viral nucleic acid in clinical specimens. 2.Isolation and identification of the virus. 3.Serological demonstration of antibody to the virus.

I- Direct Detection:
a) The electron microscope b) Fluorescent-antibody test c) Double immunodiffusion d) ELISA e) Nucleic acid probing: f) Polymerase chain reaction (PCR) Direct cytological examination i.e.cytopathology: The most important cytological changes of viral infection are the formation of syncytia or multinucleated giant cells and the detection of inclusion bodies.

2. Isolation and identification of Viruses:

Isolation is a widely used method of diagnosing infection. Specimens: Body secretions or excretions or other material from lesions or sites where virus is commonly present. Three main systems are inoculated for virus isolation. 1) Tissue culture cells (TCC). 2) Chick embryo. 3) Laboratory animals.

a) Tissue culture:

Three main types of tissue culture cells (TCC) are used in virology. 1. Primary cell lines: It is a monolayer or single layer of cells. e.g. primary rabbit kidney, primary chick embryo fibroblasts. 2. Semi-continuous cell lines: The cells are usually fibroblasts derived from embryonic tissues, e.g. human diploid cell lines, they have a diploid number of chromosomes. There is a rapid growth rate and the cells can be subcultured up to about 50 passages in culture. 3. Continuous cell lines: Usually derived from malignant or cancerous tissue, e.g. Hela cells, derived from a cervical cancer and HEp-2 cells derived from a .human epithelial carcinoma, the chromosomes are heteroploid .There is rapid growth rate and the cells can be subcultured indefinitely Virus growth is recognized by development of: 1.CPE (Cytopathic effect): i.e. rounding, shrinkage ,ballooning and syncytia or multinucleated giant cells may occur . 2.Haemadsorption: Seen with hemagglutinating viruses which adhere to the infected cells due to the formation of haemagglutinin. 3.Haemagglutination: Appearance of haemagglutinin in the cell culture fluid to detect the presence of a haemagglutinating virus. 4.Immunofluorescence. 5.Interference: In case of viruses which do not produce CPE (e.g. rubella V.), the cells appear normal but no longer susceptible to superinfection with CPE-producing virus. 6.Detection of virus specific N.A.: as PCR. Virus Identification: For cytopathic viruses; the most widely used test for identifying virus isolated is the neutralization test. 1.Neutralization tests can be done with standard antiviral serum is tested with the unknown virus, it neutralizes infectivity and therefore prevents the usual CPE produced by the virus. 2.Haemagglutinating viruses are identified by testing for neutralization by standard antiserum which will inhibit haemagglutination (HI).

b) Chick Embryo. c) Laboratory Animals.

3. Serological Diagnosis:
Infection is diagnosed by the demonstration of the development of virus antibody. A diagnosis of recent infection depends on the following criteria. 1.Detection of IgM: the earliest antibody to appear. 2.Rising titer: Increase in the level of virus antibody at least four fold over the course of infection from acute to convalescence. 3.High stationary titer: If the titer of antibody is considerably higher than that found in the general population. Some tests used in serology:

1. Immunofluorescence:
Virus specific antibody is detected usually by the indirect technique.

2. Enzyme Linked Immunosorbent Assay (ELISA): The indirect technique for detection of antibodies is used. 3. Haemagglutination-inhibition test:
Many viruses haemagglutinate erythrocytes but virus antibody blocks this. Antibody can be detected in patients serum by inhibition of virus haemagglutination.

4. Neutralization tests:
Antibody prevents virus infection of cells. Antibody can be detected by neutralization of the biologic effect of the virus e.g. CPE.

IMMUNOBLOTTING TECHNIQUES
These techniques recognize the components of the antibody response. Examples: Western blot (for detection of HIV antibodies) RIBA (recombinant immunoblot assay) (for detection of HCV antibodies).