Methods of study of viruses

For isolation and cultivation:

• Embryonated eggs
  
• Animals and Plants
  
• Tissue culture

For detection, identification and diagnosis:

a) Tissue culture methods

b) Physical methods

c) Serological methods

d) Immunological methods

e) Others and molecular biology

1. Eggs

• First method used for virus cultivation
  
• Inconvenient
  
• Safety in handling animals
  
• Mainly replaced by cell culture except when:
  
• Virus has no known host in vitro
  
• Study of viral pathogenesis in a whole host
  

Some viruses will replicate in the living tissues of developing embryonated hens eggs, such as influenza virus. Egg-adapted strains of influenza virus replicate well in eggs & very high virus titres can be obtained. This method has proved to be highly effective for the isolation & culture of many (but not all) viruses.

http://chickscope.beckman.uiuc.edu/resources/egg_to_chick/figures/figure11.gif

2. Animals

Creation of transgenic animals & plants by means of the insertion into the DNA of the experimental organism of all or part of the virus genome, resulting in expression in the somatic cells (and sometimes in the cells of the germ line) of virus mRNA & proteins.

Some animal s that are used:

http://www.scanbur.eu/images/products/Lab_animals_sprague_dawley_.jpg

http://wwwimage.cbsnews.com/images/2004/05/31/image620404x.jpg

3. Plants

Tobacco Mosaic virus

Virus plaque leaves to determine virus number

http://www.apsnet.org/online/feature/tobacco/image/tobacco1.jpg

4. Tissue culture

- Cell tissue culture

- Cells grown in vitro

- Primary cell culture

- Continuous cell lines:

• derived from primary cell lines
  
• transformed/ cancerous cells
  
• polyploid or multiploid
  
• can be sub cultured indefinitely- theoretically
  
• method of choice to cultivate virus

Some viruses do not grow in vitro

http://www.nordiqc.org/Run-17-B1/Assessment/Image/NQCB1_HER-2/NQCB1-HER2

(A) Tissue culture method for detection

• Cytopathic Effect (CPE)
  
• Plaque Assay
  

Cytopathic Effect (CPE)

The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the host cell due to infection are known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell rounding, disorientation, swelling or shrinking, death, detachment from the surface, etc.

http://www.medscape.com/content/2004/00/47/13/471342/art-eid471342.fig1.gif

Plaque Assay

What?

• Observing cell death in infected cell culture
  
• One virus infects one cell and spreads to surrounding cells
  
• Higher accuracy at lower concentration
  
• Features
  
• Very time-consuming
  
• Very simple method
  
• Only works for viruses that affect monolayer
  
• Only works for viruses that cause cell lysis
  
• Uses principle of one virus on the monolayer produces one plaque
  

http://www.sigmaaldrich.com

Virus count features

• Counts only viable virus
  
• Must know culture conditions for the virus studied
  
• Useful for samples with very low virus counts
  
• Requires time for incubation
  

(B)Physical methods

• X-ray crystallography
  
• Electron microscopy
  
• Ultracentrifugation
  

(C)Serological / immunological methods

• Haemagglutination(HA)
  
• Haemagglutination Inhibition(HI)
  
• Virus neutralisation
  
• Complement fixation
  
• Immunostaining
  
• Immunoprecipitation/immunoblot
  
• ELISA
  

Haemagglutination

http://www.microbiologybytes.com/introduction/graphics/haem.jpg

Influenza and other viruses

Two spike proteins( neuraminidase and haemagglutinin)

Complement fixation

• Mediated by antibody
  
• Antibody binds to antigen
  
• Complement cascade of molecules in blood serum initiated, causing lysis of infected cell or pathogen
  

http://www.dshs.state.tx.us/LAB/images/cf_test2.gif

(D)Immunofluorescence

• Antibody tagged with fluorescent dye
  
• Antibody attached specifically to antigen
  
• View specimen under exciting light
  
• Fluorescent microscope
  

Immunoprecipitation

http://www.molecularstation.com/images/immunoprecipitation.gif

Immunoblot

http://www.teagasc.ie/research/reports/dairyproduction/5067/image01.gif

ELISA

What?

• Enzyme –linked immunosorbant assay
  
• Antibody to detect antigen
  
• Antibody labelled with indicator
  
• Colour reaction
  
• System requires one molecule to be attached to solid surface
  

http://microvet.arizona.edu/Courses/MIC419/ToolBox/ELISA2.gif

molecular virology

• study genome organisation
  
• expression of viral genome
  
• replication of genome and progeny virus
  
• molecular basis of viral replication cycle
  

(E)Molecular biology & Others

Analysis of viral proteins

• PAGE/SDS PAGE
  
• Western blot
  
• Protein sequencing
  
• x- ray crystallography
  

Analysis of viral genome

• Agarose gels
  
• Restriction analysis
  
• Sequencing
  
• Southern blot
  
• Northern blot
  
• PCR/RT-PCR

How does PCR work?

Cycles of 3 steps repeated again and again

Step1: heatingà separate double stranded DNA

Step 2: coolingà primers bind

Step 3: increase temperatureàpolymerase work