4 - Concentration and purification of viruses

Question 1

Aim

Answer 1

Virus analysis needs clean cultures

Question 2

Steps

Answer 2

1. Isolate virus
2. Plaque purification
3. Viral strain
4. Propagation of identical viruses in large scale

Propagation = formering på norsk

Question 3

Release of viruses from infected cells

Answer 3

1. Mechanical method: Freeze-thawing
2. Sonication (Heat generation, virus protein may damage)
3. Detergents (for NA investigation)

Question 4

Purification

Answer 4

• Centrifugation
  
  • ​Sedimentation of cells
• Filtration
  
  • ​Removes larger particles than virus (eg. bacteria)
  • 450nm filter

Question 5

Concentration

Answer 5

Method depends on viral resistance

1. Precipitates
   
   • ​In ammonium Sulphate, Ethanol etc.
   • Resolve in buffer
2. Adsorption
   
   • ​Aluminium hydroxide
   • Non-specific chromatography
3. Ultrafiltration
   
   • ​Hydrostatic pressure
   • Pore sizes are smaller than the diameters of viruses
4. Dialysis
   
   • Osmotic pressure
   • Through a semi permable membrane
5. Pelletisation
   
   • ​Viron sedimentation
   • In ultracentrifugate

Question 6

Methods of concentration and purification

Answer 6

1. Affinity chromatography
   
   • ​Virus-specific antibodies are bound to the chromatography column matrix
   • Adsorption of virus
   • Rinsing
   • Elution with buffer (pH change)
2. Density gradient ultracentrifugation
   
   • Stokes laws (1-2):
     
     • Sedimentation rate is proportional with the size of the particle and with the difference between the density of the particle and the density of its environment
     • Rate zonal technique:
       
       • Based on sedimentation rate, which are less than the virus (stoke 1)
     • Isopicnic technique:
       
       • Based on density differences
       • Viruses will sink as deep as their buoyant density is equal to the environment’s density (Stoke 2)
   • Virion buoyant density
     
     • Nucleic acid/protein/lipid ratio
     • Incomplete virions are lighter
   • Preparative ultracentrifugation: for purfication
   • Analytic ultracentrifugation: for measuring buoyant density
   • Virus-specific zones are collected and purified by dialysis

Question 7

What should be included in the topic?

Answer 7

1. Aim
2. Steps
3. Release of virus from the infected cell
4. Purification
5. Concentration
6. Methods of concentration and purification