1. BTY328: Viruses Dr William Stafford wstafford@uwc.ac.za
Viral isolation and identification
Diagnosis of Viral Infection

2. Overview of methods to identify virus
Histological and cellular changes (Cytopathic effects, haemadsorption)
Formation of plaques
Serological methods (IF, ELISA)‏
Electron microscopy
DNA molecular methods (PCR, hybridisation)

3. Cytopathic Effect‏
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)‏

4. 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.)‏

5. Plaque Assays

6. Plaque assay: method Titer = 1 x 107 pfu/ml 1:100 1:10 10-2 10-3 10-4
10-5
10-6
10-7
virus
serial dilution
plate 1 ml
plaques
100 10 1
(1000)‏
(100,000)‏
(10,000)‏
Titer = 1 x 107 pfu/ml

7. Plaque assay
Serial dilution to find viral titre: With and without (+/-) IBT antiviral drug
Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Fig. 2-5

8. Serological Methods

9. ELISA: HIV detection
Microplate ELISA for HIV antibody: colored wells indicate reactivity

10. Western Blot HIV-1 Western Blot Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive

11. mix with red blood cells
Hemagglutination assay
1:8
1:2
1:2
1:2
1:2
1:2
virus
serial dilution 8 16 32 64
128
256
mix with red blood cells
side view
top view
Titer = 32 HA units/ml

12. Haemagglutination assay
From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig

13. Hemagglutination assay: influenza virus
Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity. (From Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Fig. 2-8)‏

14. Immunofluorescense
HSV-infected epithelial cells from skin lesion. (Source: Virology Laboratory, Yale-New Haven Hospital)‏
Positive immunofluorescence test for rabies virus antigen. (Source: CDC)‏

15. Immune Electron Microscopy
Either transmission or scanning electro microscopy is carried to quantify viruses in a sample and determine viral structure.
EM can be enhance by using antibodies specific for a viral antigen (the antibody is usually labelled by conjugation to gold particles)

16. Electronmicrographs Adenovirus Rotavirus |____________________|
(courtesy of Linda Stannard, University of Cape Town, S.A.)‏
|____________________|
Approx. 100nm

17. Direct particle count using electron microscopy
Direct electron microscopic particle count. An electron micrograph of a spray droplet containing 15 latex beads (spheres) and 14 vaccinia virus particles (slightly smaller, brick-shaped particles). (From Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Fig. 2-7.)‏

18. DNA Molecular Techniques
Dot-blot, Southern blot, in-situ hydridization are examples of classical techniques. depend on the use of specific DNA/RNA probes for hybridization.
PCR for specific viral genes
Whole viral genome sequencing.

19. Viral isolation: Differential centrifugation
Partial purification may be achieved by resistance to chemicals (CHCl3), enzymes (DNase, Rnase).
Viruses can also be
separated from host cells
by size selected filtration and differential centriguation
(10 000g and g).

20. Virus isolation by density gradient centrifugation
Equilibrium density gradient centrifugation and rate zonal centrifugation
Equilibrium density gradient centrifugation
and Rate zonal centrifugation separates viruses from cells and cellular components based on their size and density.
Purification of specific viruses can be achieved by affinity chromatography using antibodies directed to the virus of interest.

21. Summary: Virus identification and isolation
Main clinical diagnostic techniques
Cell culture, serology and antigen detection, nucleic acid detection
Virus culture
Cytopathic effect
Not all viruses can be cultured!
Virus quantitation
Biological in vivo methods (palque assay and LD50/ID50 for animal models)
Physical (serological assays, heamagglutination, electron microscopy)
Isolation of viruses and infectious agents by physico- chemical methods
Nature and identification of viruses, (also prions, viroids…!?)