Lab 2 - Virus titration, CPEs and identification Flashcards
(38 cards)
Reasons for virus titration
- Serology: determines antibody content
- Vaccine production: determines virion (antigen) content
- Pathogenicity tests: for experimentally infection og susceptible animals
Methods for virus titration
- Electronmicroscopic investigation-physical method
- Determination of the infective titre in cell culture-biological method
- In case of haemagglutinating viruses-haemagglutinating test
What is the electronmicroscopic investigation method for virus titration?
Counting the number of virus particles
How is determination of the infective titre in cell culture happening?
10-fold serial dilution of the virus suspension is inoculated into cell cultures, then detection of the appearance of CPE in the different dilutions
What is haemagglutinin?
Surface proteins, appearing on some viruses, and able to bind erythrocytes
What can virions with haemagglutinin do?
Agglutinate RBCs to form a lattice - haemagglutination
What is viral haemagglutination titre?
The end dilution of a virus suspension, where the haemagglutination still appears - contains 1 HA unit of viruses
What is cytopathic effects (CPEs)?
Alterations in the morphology of cells due to virus infection, therefore mainly observed in cell cultures
What is CPEs result of?
- Direct damage of viruses on cells
- Toxic effect of adsorption
- Virus proteins inhibit cellular translation
- Early proteins inhibit cellular nucleic acid transcription and replication
- Viral proteins damage the cytoplasmic membrane permeability - osmotic changes
- Cytosceleton depolymerisation, expression of fusion proteins
Main type of CPEs
-Inclusion body formation: Intranuclear inclusion bodies and intracytoplasmic inclusion bodies -Cell rounding -Syncytium formation -Lumpy cell nucleus -Cell vacuolisation -Cytolysis -Haemadsorption
What is inclusion bodies?
Virus depositions
Appear at the site of assembly of nucleocapsid
Where can inclusion bodies be observed?
In stained cells, with homogenous stainin, surrounded by a narrow light stripe
What is intranuclear inclusion bodies caused by?
DNA viruses replication in the nucleus, sometimes by RNA viruses
How can intranuclear inclusion bodies be observed when stained?
Can be basophyl, amphophyl or eosinophyl particles. In case of large number, caryomegalia and/or perichromasia can be observed
What is intracytoplasmic inclusion bodies caused by?
Usually by RNA viruses, sometimes by DNA viruses replicating in the cytoplasm
How can intracytoplasmic inclusion bodies be observed when stained?
Can be eosinophyl or rarely basophyl particles
What is Pathognomic inclusion bodies?
Negri bodies and Guarnieri bodies
What is the reason for cell rounding?
Cytosceleton depolymerisation and loss of electrolytes
How can cell rounding be observed?
- Unstained: double refraction, detachment, shrinkage
- Stained: cells is intensive stained
What is syncytium formation caused by?
Exclusively by enveloped viruses. The fusion proteins of the virus are used prim. for penetration, but also cause fusion of neighbouring cells membranes
What does syncytium formation make possible?
Intracellular spread of viruses, can hide from antibodies
What is Lumpy cell nucleus caused by?
Chromatin conglomeration, rearrangement of changed refraction
What does cell vacuolisation do?
Vacuoles are formed in the nucleus/cytoplasm of the cells
Why does cytolysis occur?
Because of damage of the cell membrane or by lisosomal enzymes
