20 - Direct demonstration of virus infections II

Question 1

Direct demostration of virus infections

Which methods should be mentioned?

Answer 1

1. Virus isolation ​(topic 19)
2. Diagnostic experimental animal infection (topic 19)
3. Electron-microscope investigation
4. Virus antigen detection
5. Viral nucleic acid demonstration

Question 2

Electron-microscope investigation

Answer 2

• Morphological recognizable virions (complete / incomplete)
• Advantage: in case of viruses not replicating in cell-cultures

Methods:

1. Ultra thin section (from organs, from the predilection sites)
   
   • Fixation (glutaraldehide)
   • Embedding (durcupan resin)
   • Tungsten-or uranium salts treatment (electron absorbent)
   • Picture: cross section of viruses
2. Negative contrast method (diluted sample e.g.: fecal)
   
   • Treatment with contrast material (phosphotungsten acid)
   • Drying onto the grid (contrast material does not bind)
   • Picture: negative contrast
3. Immune-electron microscopic method (diluted samples)
   
   • Centrifugation
   • Immune serum added to the sample → precipitation
   • Centrifugation precipitated virus in the sediment
   • Picture: negative contrast
   • It helps virus identification as well

Question 3

Virus antigen detection

Answer 3

• With specific antibodies virus identification too!
• Incomplete virions, non matured proteins are demonstrated
• Proper serum is needed:
  
  • Hyperimmune
  • Monospecific (polyclonal)
  • Monoclonal

1. Immunoflurescence test (IF)
   
   • Antigen intracellulary
   • Direct: rabies, classical swine fever
   • Indirect: very sensitive
   • Specificity →← sensitivity optimisation
2. Immunoperoxidase test (IPA, PLA)
   
   • Antigen intracellularly
   • Histology slide / cell culture
   • Spec, antibody labelled with peroxidase enzyme
   • Substrate digestion → color change
3. Complement fixation test (CF)
   
   • Antigen released off the cells
   • The hemolysin lyses the sheep red blood cells in the presence of complement (FMD)
4. Agar gel immune diffusion test (AGID)
   
   • Adenovirus
   • Bluetongue
   • EHD
5. Counter current immune electro-forezis (CIEF)
   
   • Rotavirus
   • Parvovirus
   • Adenovirus
6. Radio immuno assay (RIA)​
7. Enzyme linked immunosorbant assay (ELISA)
   
   • Direct / indirect (antigen/antibody detection)
   • Competitive (discriminating / multispecies antibody detection)
   • Sandwich (FMD antigen detection)

Question 4

Viral nucleic acid demonstration

Answer 4

• Nucleic acid hybridization method
  
  1. ​In situ hybridisation: from histological sections virus DNA
  2. Dot blot hybridisation (filter hybridisation)
     
     • Homogenized organ sample, nasal swabs
     • DNA aspecific binding
     • Heat treatment
     • Specific probe DNA adding
     • Rinsing
     • Radioactivity or enzyme-substrate colour reaction
• Polymerase chain reaction (PCR)
  
  • NA can be detected from autolysed sample - amplifies the DNA
  • Small amount of NA can be amplified
  • Needs:
    
    • Template DNA
    • Free nucleotides
    • Primers
    • TAG polymerase enzyme → heat stabile
    • Reaction mixture, Mg ions
    • Thermocycler
  • Steps:
    
    • Denaturation (94ºC)
    • Primer annealing (50-60ºC)
    • Synthesis (72ºC)
• DNS microarry test
  
  • Oligo DNA-s fixation onto a glass slide
  • DNA position data saved in computer → pathogen detected by fluorescence​

Latest methods:

• Random PCR & PEER (Primer Extension Enrichment Reaction)
  
  • Prerequisite: virus propagation in cell culture
  • Specific viral nucleic acid detection
• Proximity ligation
  
  • ​For protein detection (not DNA!)
  • Amplifies the protein
  • Visualized by Gel Electroflourescence or real time PCR