Detection, Quantification & Cultivation of Viruses Flashcards
3 Lab methods for culturing viruses
- In cell culture;
- Primary Cell culture
- Secondary Cell culture
- Continuous cell culture
- In embryonated eggs
- In Lab animals
Components of Cell culture medium
- Salt solution (usu. w/ bicarbonate buffer - needs 5% CO2 in air atmos. to maintain neutral pH)
- Glucose
- Amino Acids
- Vitamins
- Coenzymes
- Antibiotics to inhibit bacterial growth
- Serum to supply additional growth factors (10% when cells growing, 2% to maintain)
Types of Cell Cultures (3)
Define Cell strain
- Primary Cell Cultures: Cells directly derived from tissue. Can be maintained for a few days in original culture vessel before they start to die or need to be subcultured.
- Diploid chromosome no. (same as parents)
- Passaged primary cells (Secondary cell cultures): Cells derived by re-trysination and re-culture in fresh medium of successfully grown primary cells.
- can subculture for few times @ weekly intervals, but finite life span
- also have diploid chromosome number
- Immortal Cell Lines: est. when cell culture is successfully subcultured many times
- can be subcultured indefinitely
- Potentially oncogenic & need lots of testing before used. Usu. originate from tumor tissue or by transformation of diploid cell strain
- usu. has little resemblance to cell of origin & varying degrees of chromosomal aberrations (Aneuploid chromosome no.)
Cell Strain: characteristics cloned from single cell and has specific features
Obtaining Primary cell cultures
- Obtain fresh, sterile embryonic tissue
- Dissociate cells physically and chemically (so there are gaps in culture but cells not destroyed too much)
- Add these cells to an appropriate medium
Explain & Define Passaging
Passaging done because it may take several days for sufficient virus to be produced in culture and for CPE to develop. Cells may age and die - may need to freeze and thaw original infected cultures when they begin aging, inoculate this material onto fresh cultures and keep doing so until CPE develops.
How to Passage Cultured Cells
*once primary cells have covered the cell culture flask = confluent.
To passage:
1. Sterilely remove medium
2. Rinse cells in sterile isotonic salt soln
3. Trypsinise the cells (w/ trypsin-EDTA)
4. Resuspend cells in fresh sterile medium
5. Estimate the cell concentration and % viable cells
6. Seed new cell culture flasks and incubate
-As soon as cells have been passaged, they = secondary cells OR cell strains
Senesence, Hayflick Limit and Transformation
Senesence: Normal cultured cells eventually die. Limit is usually 20 - 100 divisions (Called their Hayflick Limit)
Transformation: when cultured cell strains can divide without limit - sometimes happens spontaneously, most of the time induced by chemical, genetic or viral mutagens. (When infected cells assume properties resembling the properties of tumor cells)
Detecting Viral Growth in Cell Culture (4)
- Cytopathic Effect
- Immunofluorescence
- Haemadsorption
- Interference
Cytopathic Effect
*Living cells usu. stick to bottom of plastic flask - damag to the cell = cytopathic effect
- Lysis & death of cell
- Syncytial cell formation (fusing cells w/ loss of membrane - >syncytia are large, multinucleated host cells)
- inclusion body formation (like a footprint of the virus - high conc of viral components (virus bricks))
- Persistence of virus in cell w/ no overt effect (latent)
- e.g. Endogenous retroviruses, porcine circovirus - Transformation: the production of cells w/ characteristics of tumor cells
Haemadsorption & Interference
Haemadsorption: infected cells w/ certain viruses can adsorb RBC - occurs when viral haemagglutnin incorp. into plasma membrane of cell during replication (assoc. w/ budding - only produced in viruses that mature in this way).
-Useful for detecting replication of non-cytopathic viruses & early infection of cytopathic viruses
Interference: Multiplication of one virus may inhibit multiplication of another also infecting the cell
Embryonated Eggs - detecting Viral growth
Virus growth recognised by:
-Death of embryo (e.g. influenza)
-Haemorrhagic lesions in embryo
-dwarfing (e.g. IBV)
-Pocks or plagues on membranes (esp. poxvirus)
Haemagglutination of RBC by allantoic fluid (e.g. influenza)
Lab Animals
- seldom used for routine cultivation of viruses
- suckling mice are used for isolation of some arboviruses and for rabies virus
Quantification of Viruses: 3 Traditional Methods
- Plaque Assay
- Tissue Culture 50% Infectious Dose
- Haemagglutination
Plaque Assay Method
- Make dilutions of virus (1 dilution into each well)
- Each dilution inoculated into culture dish
- Media added that incorporates agar (agar restricts spread of virus from infected cells)
- After few days stain w/ a vital stain
- Each plaque (non-staining area) represents the CPE caused by one infectious virus particle (Becomes visible to the naked eye)
Estimation of TCID50 Method
-2 statistical methods used
- Serial dilutions of a virus suspension made & each dilution is used to infect cell culture monolayers
- Usu. 5 separate cultures infected w/ each dilution
- Cultures then examined for evidence of viral infection (i.e. CPE) & titre of virus expressed as highest dilution of the virus which will produce 50% of inoculated cell cultures
*relies on statistical approximation (either spearman - Karber or Reed-Muench methods) - but can be used for viruses that do not cause plaque formation
