Cultivation of Viruses Flashcards
What are 3 reasons we would want to cultivate a virus?
Isolation and identification
Scientific and diagnostic purposes
Vaccine production
What are 3 methods of cultivation?
Animal inoculation
Inoculation into an embryonated egg
Cell culture
When would we use animal cultivation?
virus cannot grow efficiently in cell culture
produce vaccine – inoculation of rabbit with hog cholera –> virus replication in liver –> livers used to produce vaccine
What are 3 characteristic symptoms of animals used for viral cultivation?
paralysis
tumors
death
When cultivating in embryonated eggs, what eggs should be used?
Chicken eggs at 7-10 days post-fertilization
What are 5 routes of inoculation in embryonated egg cultivation? Describe signs of growth in each area
- yolk sac – dwarfing of embryo
- Chorioallantoic membrane – pocks
- Amniotic – hemagglutination
- Allantoic – hemagglutination
- Intravenous – death of embryo
What are 3 types of cell culture used for viral cultivation?
- Primary cell culture
- Finite or diploid cell lines
- Continuous cell lines
What is the major difference between the three types of cell culture?
of passages (multiplications) + magnitude of culture medium
- Primary – few passages + high culture medium
- Finite or diploid cell line – up to 60 passages + intermediate culture medium
- Continuous cell lines – infinite passages + low culture medium
What cells are used for each type of cell culture medium?
Primary - kidney cells, hepatocytes, and neurons
Finite or diploid cell lines - fibroblasts from lung
Continuous cell lines - Hela cell from carcinoma of cervix
What are some ingredients of cell culture medium?
Salts, AA, vitamins, nucleosides, minerals, organic supplements, glucose
What is added to the medium and why? Will this be beneficial?
Serum – provides protein, hormones (GH, insulin, hydrocortisone), trace elements (Fe, Cu, Zn, Se), intermediary metabolits, other nutrients
Bovine serum may contain viruses (BVD) = possible contamination.
Serum may come with BVD - or + result
What is the sequelae (6) for viral inoculation of cell cultures? (Hint: what will appear with successful virus inoculation?)
- General cell death
- Plaque formation
- formation of giant or syncytial cells through cell fusion
- Formation of inclusion bodies (nuclear or cyto)
- Cell transformation
- Persistent infection
Cell damage by virus is due to 5 factors?
- Accumulation of viral structural components
- Formation of virion aggregates w/in the cell
- Shutdown of cellular protein synthesis
- Shutdown of cellular NA synthesis
- Some viral gene products can induce apoptosis
What are 6 ways inclusion bodies present in virus infected cells?
- intracyto acidophilic
- intranuc acidophilic (cell fusion - synctium)
- perinuclear intracyto acidophilic
- intracyto acidohilic (Negri bodies)
- intranuc basophilic
- intracyto + intranuc acidophilic (synctium)
What is the prep steps for primary cell culture?
- remove organ
- wash and mince in sterile buffered saline
- transfer to trypsin solution – disaggregation. Filter through cheesecloth to remove large pieces
- sediment cells - low speed centrifugation
- discard supernatant and resuspend cells in warm growth medium
