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BTY328: Viruses Dr William Stafford wstafford@uwc.ac.za

BTY328: Viruses Dr William Stafford wstafford@uwc.ac.za. Viral isolation and identification Diagnosis of Viral Infection. Overview of methods to identify virus. Histological and cellular changes (Cytopathic effects, haemadsorption) Formation of plaques Serological methods (IF, ELISA) ‏

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BTY328: Viruses Dr William Stafford wstafford@uwc.ac.za

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  1. BTY328: VirusesDr William Staffordwstafford@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. 1:100 1:10 1:10 1:10 1:10 1:10 virus serial dilution 10-2 10-3 10-4 10-5 10-6 10-7 plate 1 ml plaques (100,000)‏ (10,000)‏ (1000)‏ 100 10 1 Titer = 1 x 107 pfu/ml Plaque assay: method

  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. 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. 51-6.

  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 filtrationand differential centriguation • (10 000g and 10000 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…!?)

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