LABORATORY DIAGNOSIS OF VIRAL INFECTIONS.

Prof. Abbas Hayat

Virus infections are diagnosed by:

1.SEROLOGY, i.e. demonstration of virus antibody.

2. Isolation of virus.

3. Direct demonstration of virus or antigen in material from the patient.

1. SEROLOGYDepends on detection of Virus Ab. Widely

used method.

Detection of Virus AntibodyVirus Abs are common in healthy human populations and can remain at high level

for many years.

Diagnosis of recent infection depends on the following criteria:

1.Detection of IgM: Earliest Ab to appear, only present in recent inf.

Detected by using specific antihuman IgM and test serum against virus Ag by ELISA or Immunofluorescence.

2. Rising Titer: Four fold increase over the course of infection from the acute phase into the convalescence.

Titer is the highest dilution of an antiserum at which activity is demonstrated: usually expressed as the reciprocal of the antiserum dilution i.e. 60 rather than 1/60.

3. High Stationary Titer. Unreliable but if considerably higher than in general population, recent infection with virus can be assumed.

Test Use d in Serology:

1. Enzyme-linked Immunoabsorbent assay (ELISA)Widely used, can detect anti-human IgM (or anti-IgG) antibody is used to detect specific IgM (or IgG) in the serum under test.

Labeled human antibody is used to detect virus Ab . The label is an enzyme which reacts with suitable substrate to produce visible change. Enzyme substrates most often used are:

a.Horseradish peroxidase and hydrogen peroxide.

b.Alkaline phosphatase

Virus + Patient serumAdd enzyme-labeled anti-human IgM antiserum

Incubate and then Add Substrate

Measure reaction by color intensity in optical density reader.Calculates as positive or negative reaction by comparison with controls

2. Radioimmunoassay (RIA):

Most sensitive technique, similar to ELISA but the detecting anti-human antibody is tagged with an isotope most often I 125 . Radioisotopes require special Labs for their handling.

Antibody Capture Tests: Both ELISA and RIA can be made more sensitive and specific by capturing patients IgM, reacting it with virus and then by adding labeled monoclonal antiviral antibody.Anti-igM antiserum fixed to well of plastic plate + Patient’s serum

Add virus

Add I125 –labeled Mouse monoclonal antivirus antibody

Count radioactivity in counter

3. Complement Fixation Test: Virus antibody is detected by fixation of added complement when the Ab combines with virus Ag.

Fixation rendered visible by latter addition of sheep erythrocytes sensitized by addition of anti-erythrocyte antibody.

If virus antibody present, complement is fixed and the sheep red cells do not haemolysed.If no virus Ab present, the complement lyses the sensitized erythrocytes.

4. Immunofluorescence: Virus specific Ab detected by indirect or sandwich technique. Patient’s serum added to spots of virus infected cells on microscopic slides.

After washing virus antibody detected on cells by application of fluorescein-labeled anti-human IgG or IgM.

Fluorescence is detected by using a fluorescent microscope.

5. Haemagglutination Inhibition Test:

Many virus haemagglutinate erythrocytes but virus antibody blocks this.

Ab. can be detected in patients by inhibition of virus Haemagglutination.

6. Neutralization:

Antibody prevents virus infection of cells.

Ab can be detected by neutralization of cytopathic effect (CPE) in tissue culture.

VIRUS ISOLATION:Virus isolation requires use of living cells. There are

three main systems.1. Tissue culture.

2. Chick embryo (rarely used now, useful for preparation of bulk virus, e.g. for antigen or vaccine production.)

3. Laboratory animals. (some can only be isolated by inoculation of laboratory animals, usually mice, after inoculation the animal are observed for signs of disease or death. Viruses are detected by testing for neutralization of their pathogenicity for animals by standard anti-viral sera.

Tissue Culture: Tissue culture is cell culture in vitro and consists of monolayer of actively metabolizing cells adherent to a glass or plastic surface in a test tube, Petri plate or on one side of a bottle.

There are three main types of tissue cultures.

a. Primary Cultures:

Short-lived but susceptible to high range of viruses, little cell division, although one subculture can be done. Cells die in two or three weeks such as monkey kidney.

b. Semi-Continuous Cell Strains:

Established from human embryo lung:Easy to maintain, can be subculture for 30-40 passages before the cells die off.

Susceptible to wide range of viruses.

c. Continuous Cell Lines: Can be subcultured indefinitely, easy to maintain, generally susceptible to fewer viruses. HeLa (derived from human cervical cancer) is the most widely known.

Virus growth is recognized by:

1.CPE or cytopathic effect: Virus kills the cells which round up and fall off the glass. Some viruses cause cell fusion and their growth is recognized by appearance of syncytia.

2. Haemadsorption: Added erythrocytes adhere to the surface of infected cells with haemagglutinating viruses.

3. Immunofluorescence: Infected cells are detected by fluorescence.

DIRECT DEMONSTRATION OF VIRUSES:

Becoming a widely used--fast method of virus diagnosis. Virus or virus antigen is detected in lesions, the fluids, tissues or excretions from the patient and the result can be obtained within an hour or two of receipt of the specimen. There are 3 main techniques :

1. Serological: Preferably with monoclonal antiviral

antibody. The most popular method is Immunofluorescence;

ELISA is also being used for this; especially useful for rapid diagnosis of respiratory virus infection.

2. Electron Microscopy:

Virus particles are detected and identified on the basis of their morphology. Widely used for detection of fecal viruses that

causes gastroenteritis.

3. Probes:

Radioactive virus DNA can be used to detect virus genome or mRNA in tissues or fluids by molecular hybridization. The PCR technique provides a powerful tool for amplification of virus nucleic acid in tissues, cells, body fluids etc.

Inclusion bodies:

are virus induced masses seen in nucleus or cytoplasm of infected cells. e.g. Negri bodies , as in rabies.

DETECTION ANTIBODY:

Another detection method using antibody from patients is the WESTERN BLOT method of

separating antigens (purified viral proteins) by charge and size and then using the patient’s serum to look for specific antigen/antibody

complexes.