21 - Titration of viruses

Question 1

Aim

Answer 1

• To standarize the virus suspension, so infective viruses of a known concentration are added to a biological test
• Used for:
  • Diagnostics (VNT, HAI)
  • Vaccine production
  • Experimental animal infection (challenge)

Question 2

Which methods can be used?

Answer 2

• Physical assay
  
  • ​Direct particle counting - EM
  • Haemagglutination
• Biological assay:
  
  • ​Determination of the infective titre (endpoint method)
  • Plaque assay
  • Focus assay
  • Pock formation

Question 3

End point dilution

Answer 3

• Inefective titer
• In vitro propagation and identification

Method:

• Serial tenfold dilution of the virus suspension
• Inoculation of the cell-cultures with each dilution
• Incubation (period is characteristic to the virus)
• If there are CPE in 50% of the inoculated cell-cultures = inefective titer
• The highest dilution of the virus, which cause CPE in the 50% in the inoculated cell cultures, characteristic to the virus suspension
• If CPE in 50% of cultures = 1 Tissue culture infective dose (TCID/50ml)
• Titration in embryonated eggs determines EID₅₀: pock assay
• Titration in experimental animals determines LD₅₀

Methods of calculation:

• Reed-Muench method: most commonly used
• Spearman-Kärber method: easier, no need of lot of numbers, only the data of dilution which is the next higher dilution of the dilution with 100% response (CPE)

Question 4

Plaque counting

Answer 4

• Inefective titer
• Serial tenfold dilution of the certain virus suspension inoculation of the cell cultures from each dilution
• Adsorption: 1 hour, 37°C
• Covering with semisolid maintenance: agar or CMC
  
  • Incubation characteristic to the virus
  • Local CPE in the inoculated cell-cultures
  • Staining with vital stain:
    
    • Gentian purple
    • Evans blue
    • Janus green

Evaluation:

• The concentration of the virus suspension is given in plaque forming units = PFU / ml

Method:

• Higher dilution
• Can count plaques
  
  • 1 plaque = 1 virus
    *

Question 5

Hemagglutinating titer

Answer 5

• Hemagglutination titer = the highest dilution of the virus suspension, in which we can observe hemagglutination
• Performed on micro heagglutinating plates (Takatsy plates)

Question 6

Hemagglutinating spectrum

Answer 6

The different hemagglutinating viruses have different hemagglutinating spectrum

Picture: example of hemagglutinating spectrum

Question 7

Comparison of the assay

Answer 7

• The physical assays count the number of particles regardless infectivity
• The biological assay detect only infecting viruses
  
  • Incomplete/defective particles are not detected
  • Purification may damage virions
  • Successful infection may also depend on the cell metabolic stage