Post on 03-Jan-2016
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BTY328: VirusesDr William Staffordwstafford@uwc.ac.za
Viral isolation and identificationViral isolation and identification
Diagnosis of Viral InfectionDiagnosis of Viral Infection
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)
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)
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.)
1:100
1:10 1:101:101:101:10
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
Plaque assay: method
Plaque assay
Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Fig. 2-5
Serial dilution to find viral titre: With and without (+/-) IBT antiviral drug
Western Blot
HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive
Titer = 32 HA units/ml
Hemagglutination assay1:8
1:2 1:21:21:21:2
8 16 32 64 128 256
virus
serial dilution
mix with red blood cells
side view
top view
From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6.
Haemagglutination assay
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)
Hemagglutination assay: influenza virus
Immunofluorescense
HSV-infected epithelial cells from skin lesion. (Source: Virology Laboratory, Yale-New Haven Hospital)
Positive immunofluorescence test for rabies virus antigen. (Source: CDC)
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)
Electronmicrographs
RotavirusAdenovirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)|____________________|Approx. 100nm
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.)
Direct particle count using electron microscopy
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.
Viral isolation: Differential centrifugation
Partial purification may be achieved by resistance Partial purification may be achieved by resistance to chemicals (CHCl3), enzymes (DNase, Rnase).to chemicals (CHCl3), enzymes (DNase, Rnase).
Viruses can also be separated from host cells by size selected filtrationfiltration and differential centriguatiodifferential centriguation(10 000g and 10000 g).
Virus isolation by density gradient centrifugationVirus isolation by density gradient centrifugation
Equilibrium density Equilibrium density gradient centrifugationgradient centrifugation and Rate zonal Rate zonal centrifugationcentrifugation 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.
Equilibrium density gradient centrifugationEquilibrium density gradient centrifugation and rate zonal centrifugationrate zonal centrifugation
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…!?)