viral diseases

1. LABORATORY DIAGNOSIS
OF VIRAL INFECTIONS.
Prof. Abbas Hayat

2. 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.

3. 1. SEROLOGY
Depends on detection of Virus Ab. Widely
used method.
Detection of Virus Antibody
Virus 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:

4. 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.

8. 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:

9. a.Horseradish peroxidase and hydrogen
peroxide.
b.Alkaline phosphatase
Virus + Patient serum
Add 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

10. 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.

11. 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

13. 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.

14. 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.

15. 5. Haemagglutination Inhibition Test:
Many virus haemagglutinate
erythrocytes but virus antibody
blocks this.
Ab. can be detected in patients by
inhibition of virus Haemagglutination.

16. 6. Neutralization:
Antibody prevents virus infection
of cells.
Ab can be detected by
neutralization of cytopathic effect
(CPE) in tissue culture.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

24. 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.

25. 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 :

26. 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.

27. 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.

28. 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.

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

32. 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.