9 - Virus isolation and culture Flashcards
Direct virus detection
Measurement of virus particle
- Electron microscopy
- Cell culture
- Molecular methods
- Serological methods
Indirect detection
Measurement of virus specific host immune response after infection
Electron microscopy
Does not require organism-specific reagents (unlike serological or biochemical identification)
Virus isolation
- Viruses can be isolated from infected tissues by propagation in animal or human cells, eggs or lab animals
- Cannot replicate outside of living cells
Cell culture medium
An isotonic solution of salts, glucose, vitamins, and amino acids buffered to physiologic pH (7.2-7.4
Three main kinds of cell cultures
- Primary cell cultures
- Diploid cell line
- Continuous cell lines
Primary cell cultures
- Prepared from human or animal tissues and have a life span of 5 -10 cell divisions.
- Derived from monkey kidney, human foreskin, embryos
Diploid cell line
Consists of single cell types and can divide up to 100 times, retaining diploid chromosome number. E.g. WI-38 from human embryonic lung
Continuous cell lines
- Consist of a single cell type that can be propagated indefinitely.
- Derived from a tumour or by treating cultured cells with a mutagen or transforming virus.
- Less differentiated than the cell of origin, and have abnormal chromosome numbers.
- E.g. Vero cells derived from African green monkey kidney epithelium
Cytopathic effects
Virus induced cell changes
Types of CPE
- Inclusion bodies
- Morphological alterations
Examples of morphological alterations of CPE
- Adherent cells round up and detach from the supporting surface
- Cells fuse together to form multinucleated giant cells or syncytia
- Apoptosis
Steps of PCR
- Denaturation of target DNA (98ºC)
- Annealing of primers (55ºC)
- Extension of primers with Taq polymerase (72ºC)
RT PCR
Reverse Transcriptase PCR. Template RNA is first converted to DNA
Immunofluorescence
An antibody specific for the antigen of interest is used to detect the presence of the antigen. Can be direct or indirect
Direct IFA
the antibody is labeled with a fluorophore and attaches directly to viral antigen
Indirect IFA
A second antibody, which recognises the Fc region of the first antibody (which is specific for the antigen of interest), is labeled with a flourophore
Plaque assay
Areas of killed cells in a tissue culture vessel. Only viruses that cause visible damage to cells can be assayed this way
Process of plaque assay
- 10 fold dilutions of virus stock are prepared and inoculated onto susceptible cell monolayers
- Cells are covered with gel that restricts spread of new viruses to neighbouring cells
Detection of viral antigen by haemagglutination (HA)
- Some viruses can bind to sialic acid on RBC and agglutinate the RBC
- Non agglutinated RBC settle in the bottom of the test well as well as a button, agglutinated cells form a smooth lattice
