Lecture 19. Cultivation of Viruses & the Single-Step Growth Cycle Flashcards
How are primary cell cultures prepared?
From animal organs or tissues
The cells are first extracted from tissues to break up tissue and release single cells.
They must be suspended in a liquid culture medium in a Petri dish or tissue-culture flask
Cells stop dividing due to contact inhibition
What are continuous cell cultures?
Usually originate from naturally occurring tumours
They are heteroploid cells and will divide forever
Can be passaged or sub-cultured many times (immortal)
Have lost their contact inhibition and as a result can grow in piles or lumps
What are HeLa cells?
The first continuous tissue-culture cell line
Were used to establish tissue culture as an important technology for research in cell biology, virology, and medicine
What are Cyto Pathic Effects (CPEs) of viral infections?
Distinct observable cell abnormalities/changes in the cells due to viral infection
What is the CPE of poliovirus?
Shrinking
What is the CPE of herpesviruses?
Synctium
What is Haemadorption?
Cells infected with certain viruses acquire the ability to bind to and adsorb red blood cells
What viruses can cause haemadsorption?
Influenza, parainfluenza, measles, mumps virus, picornaviruses, Haemagglutinins (HA)
What are the caveats of cell cultures?
- Relatively slow (1-3 days (HSV) to 3> wks (CMV)
- Low sensitivity
- Successful culture depends on the viability of the virus in the specimen (collection and transport of specimen)
- Cell cultures are very susceptible to bacterial contamination
- Cell culture is not applicable to a number of viruses
How can the number of viral particles be measured?
Electron microscope
What are the downsides to using an electron microscope?
Time consuming
Expensive
Requires skilled personnel
How can the number of viral proteins be measured?
Enzyme-linked immunosorbent assay (ELISA) Immunofluorescence (IMF)
How can the number of viral nucleic acid copies be measured?
Molecular biology, PCR
How can cytopathic viruses be quantified?
Plaque assay
How are plaque assays carried out?
Virus is added to monolayer of cultured cells (at a specific dilution or inoculum)
Agar is added on top (semi-solid overlay) to prevent diffusion
Infect separate plates for each dilution, cover with semi-solid agar, incubate and stain with crystal violet. Live cells stain and plaques show as cleared areas
