Virology

1. Laboratory Diagnosis
of Viral Infections

2. Three Approaches
1. Direct detection of virus
• Electron Microscopy
• Light Microscopy “Inclusion Bodies”
• Antigen detection tests
• Molecular Methods: PCR & Nucleic Acid Probes
2. Virus Isolation (Indirect detection)
• Animal inoculation
• Inoculation of eggs
• In vitro Cell Culture
3. Serology (Detection of Antibodies)

3. Electron Microscopy (EM) &
Immune Electron Microscopy
• Magnification : 50,000 - 400,000
• Mainly used for the diagnosis of viral gastroenteritis
• The sensitivity and specificity of EM is enhanced by
immune electron microscopy
Drawbacks of EM
• Expensive
• Poor sensitivity: at least 105
to 106
viruses per ml in the
sample required for visualization
• Need highly skilled observer
• Becoming less widely used due to availability of reliable
antigen detection and molecular methods.

4. Rotavirus Astroviruses
Adenovirus Norwalk-like
viruses
Transmission EM

5. Light Microscopy
• Detect inclusion bodies (IB)
• IB are collections of replicating virus
particles in the nucleus or cytoplasm of
infected cells.
• Seen in histological sections
• May be characteristic or non-specific
e.g. Negri bodies in rabies

6. Antigen Detection Tests
 Viral antigens can be detected by a wide
range of serological techniques utilizing
polyclonal or monoclonal antibodies.
 The same techniques, utilizing purified
viral antigens, can be used to detect
specific antibodies to those viruses in the
patient's serum.

7. Immunofluorescence
• Rapid: result within a few hours
• Technique is often tedious and time consuming
• Result difficult to read and interpret
• Poor sensitivity and specificity
• The quality of the specimen obtained is of utmost
importance in order for the test to work properly

8. Molecular Methods
• In recent years extensively used for "non-
cultivable" viruses
• Use in a routine diagnostic lab is increasing
• The future of viral diagnosis
• Have greatly improved the specificity of
virus diagnostic
• Used for both protein and nucleic acid

9. • Polyacrylamide gel electrophoresis (PAGE) of
protein fragments
• Western blotting
• Polymerase Chain Reaction (PCR), to amplify
specific segments of viral nucleic acid
• Southern blotting, and DNA hybridization
with labelled probes
• Sequencing of portions of the viral genome
• Restriction fragment Length Polymorphisms
(RFLP) of viral nucleic acid
Molecular Methods

10. Virus Isolation
1. Inoculation of laboratory animals
• Observing the animal for signs of disease.
• One of the earliest ways of detecting a virus
2. Inoculation of fertile hens eggs
 Animals and eggs are difficult to handle
and are rarely used
3. "in vitro" cell cultures are still used

11. 1. Primary cells - e.g. Monkey Kidney
• These are normal cells obtained from
freshly killed adult animals.
• These cells can only be passaged once or
twice
o The best cell culture systems
o Support the widest range of viruses
o Are very expensive
o Are difficult to obtain a reliable supply
Virus Isolation: Types of Cell Cultures

12. 2. Semi-continuous cells - e.g. Human
embryonic kidney and skin fibroblasts.
• Are taken from embryonic tissue
• May be passaged up to 50 times.
3. Continuous cells - e.g. HeLa, Vero, Hep2
• These are immortalized cells i.e. tumor cell lines
• May be passaged indefinitely
• The most easy to handle but
• The range of viruses supported limited
Virus Isolation: Types of Cell Cultures

13. • Cytopathic Effect (CPE)
o Specific CPE: e.g. HSV and CMV
o Non-specific CPE: e.g. enteroviruses
• Haemadsorption
o Cells acquire the ability to stick to mammalian
RBCs
o Haemadsorption is mainly used for the
detection of influenza and
parainfluenzaviruses.
Virus Isolation:
Identification of growing virus

14. CPE
HSV RSV
Enterovirus 71

15. Problems with cell culture
• Delayed results (up to 4 weeks)
• Sensitivity is often poor
• Susceptible to bacterial contamination and toxic
substances in the specimen.
• Many viruses will not grow in cell culture at all e.g.
Hepatitis B and C, Diarrheal viruses, parvovirus
 The role of cell culture is declining due to rapid
methods like antigen detection & molecular
methods
Virus Isolation:
Identification of growing virus

16.  The mainstay of viral diagnosis using
polyclonal or monoclonal antibodies
Classical Techniques
1. Complement fixation tests (CFT)
2. Haemagglutination inhibition (HAI) tests
3. Immunofluorescence techniques (IF)
4. Neutralization tests
 CFT and HAI, can only detect total antibody,
which comprises mainly IgG
 Are less sensitive
Serology

17. Newer Techniques
1. Radioimmunoassay (RIA)
2. Enzyme linked immunosorbent assay (EIA &
ELISA)
3. Particle agglutination
4. Western Blot (WB)
5. Recombinant immunoblot assay (RIBA)
 EIA and RIA, can detect specific IgM or IgG,
 EIAs and radioimmunoassays are the most
sensitive tests available
Serology

18. Primary Infection
• IgM is the first antibody to appear; is
followed by a much higher titre of IgG.
Reinfection
• The level of specific IgM either remains
the same or rises slightly
• IgG shoots up rapidly and far more
earlier than in a primary infection
Serology
Primary Infection