traditional diagnostic tools for diagnosis of viral diseases..molecular tools not included as they are more new age.

1. TRADITIONAL DIAGNOSTIC
TOOLS USED IN VIRAL DISEASES.

2. VIRAL CULTURES
- Cell cultures are derived from dispersed cells
taken from original tissue and disaggregated
by enzymatic, mechanical or chemical means.
- Cell cultures gained interest in virus isolation in
the 1950s’, largely due to the discovery that
poliovirus would proliferate in cell cultures
that were not of neutral origin.

3. VIRAL CULTURES
-Cell cultures:
i) Are convenient and less expensive than egg
and animal
ii) Are convenient for microscopic examination
for cell proliferation.
iii) Provide a desirable environment for the
detection and identification of many human
viral pathogens.

4. VIRAL CULTURES
-Virus isolation in cell cultures has been the gold
standard as a method for virus detection, but
in recent years powerful tools have been
discovered for viral detection, from
development of monoclonal antibodies to the
introduction of molecular diagnostics.
-The above modalities are sensitive and highly
specific in viral identification.

5. VIRAL CULTURES
- Cell cultures can be prepared in standard 16- by
125-mm glass or plastic round-bottom screw-cap
tube.
- Well known cell types found in virology
laboratories include: primary rhesus monkey
kidney (RhMK) cells, primary rabbit kidney cells,
human lung fibroblasts (MRC-5), human foreskin
fibroblasts, human epidermoid carcinoma cells
(HEp-2), human lung carcinoma cells (A549), and
others.

6. VIRAL CULTURES
-Once a clinical sample is obtained, its
inoculated into several cell lines, so as to
provide a suitable host for whichever virus
that may be present.
-For successful virus isolation, you need to
employ the appropriate selection, collection,
transport, and processing of clinical samples.

7. VIRAL CULTURES
-You need to collect the sample with the highest
titer, and preserve that sample while
maintaining the viral titer and infectivity.
- In general, if you are sending specimens for
virus culture, send them in virus transport
medium. Do not send dry swabs for virus
investigations – they will almost always be
discarded.

8. VIRAL CULTURES
- The sample should be kept cool(2-8 degrees
or on wet ice). This preserves the viral
infectivity and increases the virus recovery,
especially for labile viruses.
- Once the sample arrives in the lab, the liquid
medium is centrifuged. Viruses tend to remain
dispersed in the liquid while bacteria, fungi,
cells, blood, mucus and fibers move to the
bottom of the spun bottle.

9. VIRAL CULTURES
- The processed inoculum may be added to the
culture by either simply adding 0.2 ml or 0.3 ml of
the sample to each tube or by adsorption
inoculation.
- In adsorbtion inoculation, the cell culture
medium is decanted from the cell culture
monolayer.
- The inoculum is applied directly to the
monolayer. It’s incubated for 30-90 minutes in a
horizontal position at 35-37degrees.

10. VIRAL CULTURES
- Excess inoculum is discarded and fresh cell
culture medium is added.
- This process enhances adsorbtion of the viral
particles and recovery of some viruses.
- The viral tube cultures are incubated for day
to weeks, depending on the specimen and
virus

11. VIRAL CULTURES
- Microscopic examination is done daily for the
first week of incubation to maximize the
detection of viral growth and on alternate
days for the remainder of the incubation
period.

12. CYTOPATHIC EFFECTS(CPE)
- These are morphological changes on the cell
caused by viral infections.
- Some CPE can be observed in unfixed unstained
cells under low power, some require fixation and
staining to see the manifestation of the viral
infection.
- There are several types of CPE. Note however
that a virus may not conform to the norm for its
family or may produce different CPE in different
host cell types.

13. CPE Cont’d
- The rate of CPE can also be used to identify
viruses;
i) Rapid viruses- the CPE appears within 1-2
days in cultures inoculated at low multiplicity
of infection(<0.1).
ii) Slow viruses- the CPE appears within 4-
5days in cultures inoculated at low multiplicity
of infection(MOI).

14. CPE
- At a high MOI, all CPE can occur rapidly.
Therefore, decisions about the rate of CPE
should be based on the lowest MOI that
produces CPE.

15. TYPES OF CPE
a) Total destruction
- All cells in the monolayer are destroyed. They
shrink, become dense and detach(death)
from the glass within 72hours.
b) Subtotal destruction
- There is detachment(death) of some of the
cells but not all.

16. TYPES OF CPE
c) Focal degeneration
- In this case the viruses show localised areas of
infection(foci).
- This focal nature is due to the direct cell to cell
transfer rather than diffusion through the
extracellular medium.
- Eventually the entire monolayer may be
involved.

17. TYPES OF CPE
d) Swelling and clumping
- The infected cells enlarge and clump together
in ‘grape-like’ clusters.
e) Foamy degeneration(vacuolization)
- Here the virus causes production of large
and/or numerous vacuoles.
- Vacuolization is difficult to visualize without
staining.

18. TYPES OF CPE
f) Cell fusion (syncytium or polykaryon formation)
- The plasma membrane of four or more cells fuse
producing a multinucleated cell with four or more
nuclei.
- Small syncytia are seen only after staining.
- The cytoplasm needs to be examined carefully so
as to distinguish between cell fusion and cell
clumping, where each cell has its distinct plasma
membrane.

19. TYPES OF CPE
g) Inclusion bodies
- Indicates areas of the cell where nucleic acid or
viral protein are being synthesized or where
virions are being assembled.
- The areas have altered staining and cannot be
seen on live cell cultures.
- -The inclusions may be single or multiple, large or
small, round or irregular in shape, intracellular or
intracytoplasmic, eosinophilic or basophilic,
depending on the causative virus.

20. SEROLOGY ANTIBODY ANTIGEN TESTS
- Serological techniques that detect antibody
are based on the principle of adding specific
viral antigen(s) to patient serum.
- If virus-specific antibody is present in the
serum then it will bind to the antigen to form
an antigen/antibody complex.
- The complex is then detected using an
indicator system.

21. SEROLOGY ANTIBODY ANTIGEN TESTS
- These tests can be reversed to detect the presence
instead of viral antigen, such as hepatitis B surface
antigen, in the patient’s serum.
- They include:
• Complement fixation test (CFT)
• Enzyme-linked immunosorbent assays (EIA or ELISA)
• Immunofluorescence (IF)
• Latex and gel particle agglutination
• Western blot (WB) and line assays (LIA)

22. Enzyme-linked immunosorbent assays
(EIA or ELISA)
- These are the most widely used serological
assays in routine diagnostic laboratories.
- The process:
i) Antigen is attached to the base of a plastic
microtitre well (solid phase).
ii) Patient’s serum is added, if specific antibody
is present in the serum it will attach to the
antigen on the solid phase. Excess serum is
washed off.

23. ELISA
iii) Anti-human antibody coupled to an enzyme is
added to bind to this antibody/antigen complex.
Excess enzyme is washed off.
iv) An enzyme for the substrate is added. Color
change shows a positive reaction due to action of
the enzyme attached to the complex to the
substrate.
- Color change can be seen by the naked eye or
measured in a spectrophotometer.
- Intensity of the color shows how much antibody
is in the serum.

24. ELISA
- The assay can be constructed to detect either
IgM or IgG, depending upon whether the anti-
human antibody is directed to the IgM or IgG
class.
- ELISA can also be reversed to detect viral
antigens, by coating the solid phase by
antibody (mono- or polyclonal) specific for the
antigen to be tested.

25. ELISA
- Its advantages include: they are rapid(done
within 2-3hours), can be easily automated and
are objective in that they can be read using a
spectrophotometer.

26. ELISA

27. IMMUNOFLUORESENCE (IF)TESTS
- Use the same principle as ELISA.
- Can also be used to detect viral antigen or
antibody in the patients’ serum.
- The enzyme/substrate detector used however
to detect a positive reaction is fluorescein-
labelled anti-human antibody which appears
as apple-green fluorescence under a light
microscope.

28. Immunofluoresence tests
- To look for viral antigen, cells from the
patient’s secretions are fixed to a spot on the
glass slide and fluorescein-labelled
monoclonal antibody against the virus is
added.
- Note that, a mixture of these monoclonal
antibodies can be added at the same time to
detect a panel of viruses.

29. Immunofluoresence tests
- Their advantage is that they are rapid
serological tests, however interpretation is
subjective, therefore depend on operator
expertise.

30. LATEX AGGLUTINATION(LA) AND
GELATIN PARTICLE AGGLUTINATION
TEST(GPAT)
- The antigen or antibody is adsorbed on latex or
gelatin, once the patients’ serum is added, a
positive reaction is noted when there’s
agglutination of the particles.

31. Latex agglutination

32. Gelatin particle agglutination test

33. COMPLEMENT FIXATION TEST(CFT)
- Is based on the principle that an antibody/antigen
complex once formed will bind complement.
- Complement activity is measured by the capacity of
serum to lyse antibody optimally sensitized red blood
cells.
-In presence of a titrated complement level, sensitized
SRBC are lysed. When complement-activating
antibodies, IgM or IgG, are present complement is
fixed and unavailable to lyse the sensitized SRBC.
- It has been used in the past, but is complex and
insensitive, so its being replaced with ELISA.

34. HEMAGGLUTINATION AND
HEMAGLUTINATION INHIBITION
TESTS
- They detect antibodies to viruses (rubella,
influenza) that possess a haemagglutinin
antigen.
- Red cells (RBC) are chemically coupled with an
antigen. They are reacted with the patients’
serum, if the specific antibodies to the antigen
are present, clumping of the cells occurs.
- They are relatively insensitive and give non-
specific results.

35. HEMAGGLUTINATION AND
HEMAGLUTINATION INHIBITION
TESTS
- In hemagglutination inhibition tests, there’s
inhibition of agglutination of antigen coated
erythrocytes by a homologous antigen.
- Antigen is detected and its quantity
determined by the degree of inhibition of
agglutination following incubation with low
concentrations of the antibody.

36. NEUTRALIZATION TESTS
- If there are virus-specific neutralizing
antibodies in the serum, then the virus will be
neutralized and will not be able to grow in
culture.
- The patients’ serum is incubated with
antibodies of known viral specificity. This
mixture is inoculated into cell culture.

37. NEUTRALIZATION TESTS
- Presence of CPE shows that the antibodies
didn’t bind, inactivate or neutralize the virus,
but a lack of CPE shows that the antibodies
bound, inactivated or neutralized the virus.
- Its sensitive, but labour intensive and
technically demanding.

38. WESTERN BLOT
- Specific viral proteins are transferred on blotting
paper either from a gel (western blot) or
produced by recombination or peptide synthesis
(line immunoassays).
- The patient serum is added and the viral antigen
band on the blotting paper develops colour if
specific antibody to that particular antigen is
present in the serum.
- Its advantage is that its very specific and is able to
distinguish antibody directed against specific
virus proteins.