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.)

Plaque Assays

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

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

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…!?)