Virus Cultivation and Purification

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What are the 3 main ways viruses are cultured in lab?

grown in cultured cells, embryonated eggs, or lab animals.

What are the 5 reasons viruses are grown in the lab?

1. isolation and identification of viruses
2. characterization of viruses
3. pathologic examination
4. serologic tests
5. vaccine production

Who first used cell culture to isolate and grow viruses? What year?

Enders, Weller and Robins in 1949

What are the 2 types of monolayer cell culture?

primary cell culture and serially propagated cell cultures.

A primary cell culture uses what type of tissue?

tissue taken directly from human or animal, they are the best culture system for isolation and propagation of viruses, they contain multiple cell types.

Which type of cell culture is used in producing viral vaccines?

primary cell culture

How many times can you culture viruses from a primary cell culture?

5 to 20 times, so they have a limited lifespan

What are the 2 types of serially propagated cell cultures? the difference?

1. diploid cell lines - homogenous population of a single cell type from human embryos or subcultures of primary culture. can subculture 100 times before growth stops.
2. heteroploid cell lines - cell of a single type capable of indefinate propagation in vitro. derived from cancers or treating a primary cell culture with mutagenic chemical. they cannot be used in vaccine production (FDA prohibits)

What are the steps in preparing a primary cell culture?

1. mince organ and treat with a protease to separate cells.
2. wash, count and dilute cells in growth medium, let settle on flat surface.
3. incubate under 5% CO2 and about 37 degrees celcius.

What are the 3 main types of growth medium used for primary cell culture?

1. MEM (eagles minimum essential medium)
2. RPMI 1640
3. Leibovitz's (L-15) medium

What 3 things are added to the growth medium of a primary cell culture?

1. serum - 5-10% sheep, horse or fetal calf serum added to provide additional growth, must be free of Abs. Note that this is for the GROWTH medium, when growth is complete transfer to maintenance medium containing 0-2% serum.
2. pH indicator - usually phenol red, most buffered to 7.4
3. Antimicrobial agents - penicillin, streptomycin, gentamicin, mycostatin and kanamycin.

What is a shell vial?

a small borosilicate glass vial containing a coverslip, used for rapid cell culture system.

What are the steps of centrifuge enhanced inoculation?

growth medium decanted from vial and placed directly on monolayer. centrifuged at low speed for an hour. fresh culture medium added, incubated at 35-37 degrees celcius. at designated time interval can do pre-CPE detection of virus by staining with virus-specific HRP. later, examine monolayer microscopically for CPE with an inverted microscope.

Describe the co-cultivated technique.

combo of different cell types grown together, as single monolayer. it is ideal for clinical samples that may contain multiple viruses. can detect different viruses labeled with fluorochrome in the same vial.

Who developed the culture of viruses in embryonated eggs? When?

Goodpasture in 1930

Must use SPF eggs for virus culture. What are they?

specific pathogen free eggs, from a disease free flock. may not contain Abs to the virus you are culturing.

How old are the embryos in eggs used for viral culture?

ideally between 5 and 13 days old.

What are the 5 routes of viral inoculation into an embryonated egg? Describe reason for each type of inoculation and ideal age of embryo for each type.

1. yolk sac - 5 to 7 day old embryo, used for togaviruses, avian enterovirus.
2. CAM - chorioallantoic membrane, 9 to 12 day old embryo, used for poxviruses, some herpes viruses.
3. amniotic route - 7 to 13 day old embryo, used for influenza A.
4. allantoic route - 7 to 13 day old embryo, used for influenza A, Newcastle disease virus, infectious bronchitis virus.
5. intravenous route - 10 to 12 day old embryo, used for ovine orbivirus (bluetongue).

What is ideal incubation temperature and humidity for virus inoculated embryonated eggs?

between 32 and 37 degrees celcius, with 62% humidity.

What are 8 signs of viral growth in embryonated eggs?

1. stunted growth (dwarfing of embryo)
2. pocks on chorioallantoic membrane
3. urate deposits on mesonephros
4. hemorrhage and congestion
5. encephalitis (sluggish movements)
6. death of embryo
7. lesions of the extracellular membranes (edema)
8. presence of hemagglutinins in embryonic fluids

What must be absent in lab animals used for viral culture?

free of antibodies.

What are the 3 major reasons lab animals are used for viral culture?

1. used for infectious agents that cannot yet be grown in cell culture.
2. used to study pathogenic mechanisms and immune response of a virus.
3. rabbits are used in production of antisera and mice for implantation of hybridomas which secrete monoclonal antibodies for serological purposes.

What are the 2 major ways of viral purification?

1. high-speed centrifugation, relative density of viruses in solution of sucrose or CsCl is constant for closely related viruses.
2. viruses layered on top of preformed gradient solution, tube centrifuged. viruses reach equilibrium position based on density in gradient solution, show up as colored band. the band is collected.

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