Viruses, Viroids, and Prions

The Isolation, Cultivation and Identification of Viruses

Viruses must be grown in living cells. They can't be grown in culture media or on agar plates alone, they must have living cells to support their replication.

The easiest viruses to grow are bacteriophages (because the easiest cells to grow in the lab are bacteria).

Growing Bacteriophages In The Laboratory

Once viruses have replicated and been harvested the concentration of viral particles (virions) in the viral stock solution must be determined. One of the easiest ways to determine the concentration of a stock solution of bacteriophages is to use the plaque method.

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 (concentration of the stock solution) of the virus is given in plaque forming units.

 

 

Growing Animal Viruses In The Laboratory

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

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

2. Embryonated eggs can serve as substitutes for some viruses.

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

 

3. 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 appear early or late in the course of the viral infection.

Cytopathic effects may be cytocidal (cell death) or non-cytocidal.

Non-cytocidal effects include acidophilic or basophilic inclusion bodies in the nucleus, cytoplasm, or both; cell fusion, and transformation.

Cytopathic effects can be so characteristic of individual viruses that they can often be used to identify viruses.

(a) Uninfected cells in culture form a monolayer (b) Cells infected with vesicular stomatitis virus round up and pile up on top of each other

 

(a) Cytoplasmic inclusion body caused by rabies virus in brain tissue.   (b) Syncytium formed by cell fusion due to infection by measles virus.

 

Viral Identification

Serological methods

Western blotting

Cytopathic effects

Diagnostic inclusion bodies are associated with rabies virus, measles virus, vaccinia virus, smallpox virus, herpesvirus, and adenoviruses.

Molecular methods include PCR and RFLPs.

PCR was used to identify the West Nile virus and the SARS-associated coronavirus