2. River’s Postulates (Modified from
Koch’s postulates)
1. Isolate virus from diseased hosts.
2. Cultivation of virus in host cells.
3. Proof of filterability.
4. Production of a comparable disease when
the cultivated virus is used to infect
experimental animals.
5. Reisolation of the same virus from the
infected experimental animal.
6. Detection of a specific immune response to
the virus.
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3. Indications for lab diagnosis of viral infection
• If rubella is diagnosed in the first trimester of pregnancy, abortion is
recommended
• If a baby is borne of an HbsAg positive mother ,abortion is recommended
For proper management
of certain diseases
• for which antiviral chemotherapy is available (herpes viruses)
Diagnosis of diseases
caused by viruses
• For hepatitis B & HIV virus helps to prevent spread of these viruses
Screening of blood
donors
• To initiate appropriate control measures
Early detection of
epidemics
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4. 3 General approaches for Laboratory diagnosis of Viral
infections
Direct demonstration of
virus & its components
Isolation of virus
Detection of specific
antibodies
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5. Microscopy
•Examine specimen for virusesElectron Microscope
•Labeled antibody
Immuno-electron
microscopy
•Fluorescent tag bound to Fc region of AbImmunofluorescence
•Histological appearance of affected cells
•Inclusion bodiesLight microscope
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7. Inclusion bodies
• Inclusion bodies are virus-specific intracellular globular
masses which are produced during replication of virus in
host cells.
• They can be demonstrated in virus infected cells under
light microscope after fixation & staining
• Eg: Negri bodies in rabies .
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12. Cell culture
Cell Cultures are most widely used for virus isolation, there are 3
types of cell cultures:
1. Primary cells - Monkey Kidney
2. Semi-continuous cells - Human embryonic kidney and skin
fibroblasts
3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK
Primary cell culture are widely acknowledged as the best cell culture
systems available since they support the widest range of viruses. However,
they are very expensive and it is often difficult to obtain a reliable supply.
Continuous cells are the most easy to handle but the range of viruses
supported is often limited.
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13. Cytopathic Effect (1)
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.
(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)
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14. Cytopathic Effect (2)
Syncytium formation in cell
culture caused by RSV (top), and
measles virus (bottom).
(courtesy of Linda Stannard, University of Cape
Town, S.A.)
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15. Haemadsorption
Syncytial formation caused by mumps virus and
haemadsorption of erythrocytes onto the surface of the cell
sheet.
(courtesy of Linda Stannard, University of Cape Town, S.A.)
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16. Tissue culture
Tissue culture /
Explant culture
Fragments of minced
tissue can be used
as “explants”
This method is rarely
used nowadays
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17. Serology
• The development of
antibodies to different
components of the
virus is used in staging
the disease. For
example in hepatitis B
and HIV infections this
approach is used.
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18. Viral Serology
– Primary and secondary responses to viral infections
• IgM (1st exposure)
• IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.9/22/2013 18Cristi Francis
19. Serological Diagnosis
• Detection of
Immunologlublins Ig G.
Ig M Ig A
• Raise of titers Ist
sample later sample
(convalescent sample)
tested after 10 – 14
days Raise of titer is
diagnostic
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20. Serology
Criteria for diagnosing Primary Infection
• 4 fold or more increase in titre of IgG or total antibody between acute
and convalescent sera
• Presence of IgM
• Seroconversion
• A single high titre of IgG (or total antibody) - very unreliable
Criteria for diagnosing Reinfection
• fold or more increase in titre of IgG or total antibody between acute and
convalescent sera
• Absence or slight increase in IgM
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21. PCR
Each cycle doubles the copy number of the target
A target DNA sequence can be
amplified to the point where
it can be readily identified
using labelled probes in a
hybridisation assay
• The technique is used for the
diagnosis of infections caused
by HIV , HPV , Herpes simplex
virus, Hepatitis B &
C,Enterovirus,EBV ,Rubella &
Rotavirus
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22. Polymerase Chain Reaction
• Advantages of PCR:
– Extremely high sensitivity, may detect down to one viral genome per
sample volume
– Easy to set up
– Fast turnaround time
• Disadvantages of PCR
– Extremely liable to contamination
– High degree of operator skill required
– Not easy to set up a quantitative assay.
– A positive result may be difficult to interpret, especially with latent
viruses such as CMV, where any seropositive person will have virus
present in their blood irrespective whether they have disease or not.
• .
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23. Detection of specific antobodies
Micro plate ELISA for HIV antibody: colored wells indicate reactivity
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24. ELISA
Procedures
Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third
Edition . ASM Press, 2000.
Figure 5-19b
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25. ELISA
• Enzyme-Linked Immuno-Sorbant
Assays (ELISAs)
–Enzyme reacts with substrate to produce
colored product
–Very sensitive
• HIV test
– If positive twice, Western Blotting is performed next
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26. Neutralization, hemagglutination, and hemagglutination inhibition assays. In the assay shown, tenfold dilutions of serum were incubated with virus. Aliquots of the mixture
were then added to cell cultures or erythrocytes. In the absence of antibody, the virus infected the monolayer (indicated by CPE) and caused hemagglutination (i.e., formed a
gel-like suspension of erythrocytes). In the presence of the antibody, infection was blocked (neutralization), and hemagglutination was inhibited, allowing the erythrocytes to
pellet. The titer of antibody in the serum was 100. pfu, Plaque-forming units.From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6.
Antibody detection
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