Transcript BTY328: Viruses

BTY328: Viruses
Dr William Stafford
[email protected]
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)

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.)
Plaque
Assays
Plaque assay: method
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
(10,000)
(1000)
100
10
1
plate 1 ml
plaques
(100,000)
Titer = 1 x 107 pfu/ml
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
Serological Methods
ELISA: HIV detection
Microplate ELISA for HIV antibody: colored wells indicate reactivity
Western Blot
HIV-1 Western Blot





Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive
Hemagglutination assay
1:8
1:2
1:2
1:2
1:2
1:2
virus
serial dilution
8
16
32
64
mix with red
blood cells
side view
top view
Titer = 32 HA units/ml
128
256
Haemagglutination assay
From Medical Microbiology, 5th ed., Murray, Rosenthal
& Pfaller, Mosby Inc., 2005, Fig. 51-6.
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)
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
Adenovirus
Rotavirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)
|____________________|
Approx. 100nm
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, brickshaped particles). (From Fields Virology, 4th ed, Knipe & Howley, eds, Lippincott Williams & Wilkins, 2001, Fig. 2-7.)
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 to
chemicals (CHCl3), enzymes (DNase, Rnase).

Viruses can also be
separated from host cells
by size selected filtration
and differential centriguation
(10 000g and 10000 g).

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.
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 physicochemical methods
• Nature and identification of viruses, (also prions,
viroids…!?)