Cultivation & Detection of Viruses Flashcards
Viruses cannot grow in standard microbiological media as they cannot replicate by themselves
Cultured inside living host cells
Three types of media for culturing viruses
- Media consisting of mature organisms (bacteria, plants or animals)
- Embryonated (fertilized) eggs
- Cell cultures
Culturing in bacteria
Most of our knowledge of viral replication has been derived from research on bacteriophages (easy to
culture)
Phages grown in bacteria in liquid cultures or on agar plates
Mix bacteria and phages with liquid warm nutrient agar and poured in a thin layer across the surface of an agar plate
Plaque assay technique
Lysis of bacteria produces plaques, clearings on a lawn of bacteria on the surface of agar
- Virulent phages make plaques (lytic life cycle)
- Temperate phages - lysogenic life cycle
Allows estimation of phage numbers
Culturing Viruses in Plants and Animals
(Disadvantages)
-use to study immune system ‘s response and used as diagnostic procedure, identify and isolate virus from clinical specimen
disadvantage:
-difficult and expensive to maintain
-ethical concerns
Embryonated (fertilized) Eggs
Advantages:
-Inexpensive
-free of contaminating microbes
-contain a nourishing yolk
-Virus inject into egg, the growth signed by changes or death of the embryo
-Some vaccines still prepared in chicken egg cultures → egg protein might contaminate to the vaccine
-different virus inject to and grow in different site in embryo
Cell culture
• cell isolated from an organism and grown on a medium or in broth
• become practical when antibiotics provided a way to limit the growth of contaminating bacteria + the use of biosafety cabinet
• less expensive than use living organism, no ethical concerns
• treat tissue with enzyme to separate cells, cells suspended in a solution with appopriate factors for them to grow
2 types of cell culture
Normal (primary) cells:
adhere to container, form mono-layer
Transformed cells (cancerous/ continous) :
do not grow a monolayer
Cell line
Primary cell lines
- derived from tissues or organs
- tend to die out after only a few generations passages
-Contact inhibition slows the growth of the
cells when too dense. At this point, growth can only be sustained by making a secondary culture
Continuous cell lines
-maintain indefinite number of generations
-grow regardless of cell density (no contact inhibition)
-Ex. HeLa cell line derived from Henrietta Lack
CPE
Cytopathic effect:
changes in cell morphology caused by infecting virus
CPE type (4)
- Cell death
- Rounding (swelling) or shrinkage of the infected cell
- Fusion with adjacent cells to form a syncytia
- Appearance of nuclear or cytoplasmic inclusion bodies (contain viral proteins, nucleic acids and other components)
Detection of virus growth
Plaque assay
Focus forming assays
Multiplicity of infection (MOI)
Plaque assay (3 times-invention)
In 1930s: used to study replication/accumulation of bacteriophages
In 1952, Renato Dulbecco developed the
technique for animal viruses
Dr. Trisha Barnard’s plaque assay with Zika virus and Vero cells (2018)
Plaque assay
- Based on the notion: a single virus is sufficient to produce a plaque
- Formation of plaque relies on virus infection & lysis of host cells
- Quantify the number of infections viral particles, concentration is reported as “plaque-forming units”per ml (PFU/mL)
-Minority of plaques are formed by the initial cell being infected by two or more viruses
MOI
Multiplicity of Infection: average number of infectious particles added per cell
- The ratio of the number of virus particles to the number of host cells in a given infection medium
Different than the number of infectious particles that infects each cell
-Ex. MOI of 1 implies that on an average, there is a a single host cell for a single viral particle
- This does not guarantee that all host cells will become infected
When cells are mixed with virus, some cells are uninfected, some receive one or more virions
The distribution of virus per cell is best described by the Poisson distribution
FFA
Focus forming assay
• immunostaining technique
• A variation of the plaque assay
• Useful for quantifying viruses that cause CPE → visible damage in host cells but not cell death
Host cells form clusters of infected cells (foci) that can be visualized with fluorescence or stain using antibodies against a viral antigen
• Each focus represents a single infectious unit
