15 - Cytopathic effects, plaque formation

Question 1

Cytopathic effects

Define cytopathic effect

Answer 1

Structural changes in host cells that are caused by viral invasion

Usually degenerative processes

Question 2

Cytopathic effects

Where can cytopathic effect be observed

Answer 2

Mainly in cell cultures

Question 3

Cytopathic effects

How can cytopathic effects be investigated?

Answer 3

• Usually investigated by light microscopy (100-400x magnification)
  
  • Some CPEs are visible in unstained cells
  • Staining usually gives a more detailed information (e.g. HE, giesma, etc.)

Question 4

Cytopathic effects

List different types of damage of viruses on cells

Answer 4

1. Toxic effect of adsorption (ie. adenoviruses)
2. Virus proteins inhibit cellular translation (ie. herpesvirus, poxvirus, togavirus)
3. Early proteins inhibit cellular nucleic acid transcription and replication (i.e. herpes, adenoviruses)
4. Viral proteins or virions are deposited in the cells
5. Viral proteins damage the cytoplasmic membrane permeability - osmotic changes (i.e. herpesviruses)
6. Cytosceleton depolymerisation (i.e. herpesviruses, paramyxoviruses)
7. Expression of fusion proteins (i.e. herpesviruses, paramyxoviruses)

Question 5

Cytopathic effect

General principels of CPE

Answer 5

• Not all virus infections results in CPE
  
  • The cytopathic character of a virus strain may change due to mutations (i.e. BVDV)
  • The CPE is not always connected to the pathogenicity of the viruses
    
    • I.e:
      
      • Classical Swine Fever: no CPE, but pathogen
      • Spumaviruses: obvious CPE, but orphan
• The appearance of the CPE is influenced by multiplicity of infections (MOI)
• CPE may occur independently or jointly
• Different viruses may cause similar CPE
• CPE may vary according to the host cell
  
  • CPE alone is not usually pathogenic
  • CPE is important character for the identification of the virus

Question 6

Cytopathic effects

List the types of CPE

Answer 6

• Inclusion body formation
  
  • Intranuclear inclusion bodies
  • Intracytoplasmic inclusion bodies
• Cell rounding
• Syncytium-formation
• Lumpy cell nucleus
• Cell vacuolisation
• Hemadsorption

Question 7

Cytopathic effects

Inclusion body formation

Answer 7

• At the site of assembly of nucleocapsid → virus deposition
• Seen in stained cells (of cell cultures or organ sections)
  
  • Homogenous staining
  • Surrounded by halo - shrinkage after fixating
• Intranuclear inclusion bodies:
  
  • ​In the nucleus replicating DNA viruses, eg:
    
    • Parvovirus
    • Papillomavirus
    • Polyomavirus
    • Adenovirus
    • Herpesvirus
  • Sometimes caused by RNA viruses, eg:
    
    • Orthomyxovirus
    • Paramyxovirus
    • Arterivirus
    • Bornavirus
  • Crowdy-A, Crowdy-B types depends on the state of the synthesis of macromolecules
  • Basophyl (ie. parvovirus)
  • Amphophyl (ie. adenovirus)
  • Eosinophyl (ie. herpesvirus)
• Intracytoplasmic inclusion bodies:
  
  • ​Usually caused by RNA viruses
  • DNA viruses replicating in the cytoplasm
    
    • Poxviruses, ASFV
  • Eosinophyl, rarely basophyl (poxviruses)
  • Pathognomic:
    
    • Negri bodies (rabies)
    • Guarnieri bodies (pox)

Question 8

Cytopathic effects

Cell rounding

Answer 8

• One of the most frequent CPE
• Cytosceleton depolymerisation
• Loss of electrolytes
• Unstained: double refraction, detachment, shrinkage
• Stained: intensive staining

Question 9

Cytopathic effects

Syncytium-formation

Answer 9

• Caused by enveloped viruses:
  
  • Alphaherpesvirus
  • Paramyxovirus
  • Pneumovirus
  • Coronavirus
• Fusion proteins (F) on the surface of the virus
  
  • Primarily for viral penetration
  • Expressed on the cytoplasmic membrane of the cell
  • Fusion of infected and non-infected cell membranes
    
    • → membrane tunnels → intracellular spread → hidden from the antibodies
  • Giant cells with many nuclei: polykaryocytes, syncytia
  • In tissue sections multinucleated giant cells may be seen
    
    • Usually lymphatic cells
    • Results of impaired cellular division

Question 10

Cytopathic effects

Lumpy cell nucleus

Answer 10

• Chromatin conglomeration, rearrangement
• Changed refraction (parvo)
• Nucleus lumpy disintegration
  
  • Equine herpes virus (EHV-1) in the liver of foals
  • African swine fever virus (ASFV) in lymphatic system

Question 11

Cytopathic effects

Cell vacuolisation

Answer 11

• Vacuoles are formed in the cells
  
  • In the nucleus (eg. by adenoviruses)
  • In the cytoplasm (eg. by flaviviruses, herpesviruses, retroviruses)

Question 12

Cytopathic effects

Hemadosrption

Answer 12

• Viral hemagglutinin is expressed on the surface of the infected cells
• RBCs are added into the culturing medium, and after 30 minutes incubation unbound RBCs are washed away
• Infected cells are capturing RBCs
• Diagnostic value: paramyxoviruses, ASF

Question 13

Cytopathic effects

The appearance of CPEs

Answer 13

• Diffuse: all around in the cell culture
• Local/focal: plaques

Question 14

Cytopathic effects

Appearance of CPEs: plaques

Answer 14

• Local/focal: plaques
• Certain viruses more frequently appear in plaques
• In lower MOI more frequently plaques are formed
• Syncytia are seen as plaques
• Facilitation of plaque formation:
  
  • Supplementing the maintenance fluid with agar, carboxymethyl cellulose or metrizamide
• Advantages of plaques:
  
  • Virus purification: subsequent passages of viruses taken from singular plaques - establishment of virus strains
  • Virus quantification: plaque counting

Question 15

Cytopathic effects

Non-specific CPEs

Answer 15

• Morphological changes in cells independently from virus infections
• Examples:
  
  • Aging of the cells
  • Alteration of the pH range of the medium
  • Alteration of the incubation’s temperature
  • Toxic components in the inoculum
  • Bacterial contamination
• Differentiation:
  
  • Compare with the uninfected control
  • Passage the isolate
  • Perform direct demostration:
    
    • PCR
    • IHC
    • ISH
    • VI