The Isolation, Cultivation and Identification of Viruses

Viruses must be grown in living cells.

The easiest viruses to grow are bacteriophages.

Growing Bacteriophages In The Laboratory

The plaque method:

Virus, bacteria, and agar mixed, plated and incubated.

After replication the virus lyses the bacteria, forming plaques, or clear zones.

Each plaque is assumed to come from a single viral particle.

The titer of the virus is given in plaque forming units.

 

Growing Animal Viruses In The Laboratory

Live animal cultures have to be used for some animal viruses.

Simian AIDS and feline AIDS provide models for studying human AIDS.

Embryonated eggs can serve as substitutes for some viruses.

Can inoculate membrane that best supports specific virus (allantoic, amniotic, chorioallantoic, or yolk sac).

Cell culture is a lot cheaper and easier to work with (contamination can be a problem however).

Primary cell lines have a short lifespan in culture – a few generations before reaching senescence.

Diploid cell lines are derived from embryos and can grow for up to 100 population doublings before senescence.

Continuous cell lines are derived from transformed cells and grow indefinitely in culture.

Hela cells – 1st continuous cell line, derived from Helen Lane (fictional name - actually named Henrietta Lacks), a cervical cancer patient who died in 1951. This is the oldest continuous cell line and was first used to culture and identify polio virus.

Transformed Cells in Culture

Viral growth can cause cytopathic effects in the cell culture. Cytopathic effects can be so characteristic of individual viruses that they can often be used to identify viruses.

Viral Identification

Serological methods

Western blotting

Cytopathic effects

Molecular methods include PCR and RFLPs.