VIROLOGY! ANY CLUE?: I-S-O-L-A-T-I-O-N...Not your usual "Lemon Tree" tune.

The title makes no sense, I know. So, why don't we continue with something more logical? =)

Now, I shall explain the methods used for isolation and cultivation :

1) Animals/Eggs

This method was the first method used for virus cultivation purposes. However, this method is inconvenient and extra attention and care is needed when handling animals. Mainly replaced cell culture except for two points :

a) The virus has no known host in vitro
For instance, Hepatitis C virus in chimpanzees and Influenza virus in chick embryo.

Diagram of mouse infection :

Reference: www.aaronlogan.com/blog/03/02/26/injection.jpg

Reference :http://www.ahwla.org.uk/site/tutorials/BVA/BVA05-Mouse/BVA05-Images/10.jpg

Here's a video about a successful intravenous dye injection in mice. The same principle applies in virus injection in mice :

Video Reference: http://www.youtube.com/watch?v=guDvNtPwDjg&NR=1

Diagram of growth of viruses in embryonated eggs :

Reference: http://www.cmbi.bjmu.edu.cn/

b) Study of viral pathogenesis in a whole host
For example, polio studies in chimpanzees.

Pathogenesis of polio virus

Humans are the only natural host of polio virus. Polio viruses have a tropism for the epithelial cells lining the alimentary tract and for cells of the central nervous system. They attach to a specific receptor on these cells, which in humans is encoded by a gene on chromosome 19. Polio virus infection is quite common in nonimmunized individuals, but only about 1 percent of these cases progress to the paralytic form of the disease.

The histocompatibility antigens HLA-3 and HLA-7 are believed to be highly associated with an increased risk of paralysis. Primary replication of polio virus takes place in the oropharyngeal and intestinal mucosa (the alimentary phase).

From here, the virus spreads to the tonsils and Peyer's patches of the ileum and to deep cervical and mesenteric nodes, where it multiplies abundantly (the lymphatic phase). Subsequently, the virus is carried by the bloodstream to various internal organs and regional lymph nodes (the viremic phase).

In most cases, no further virus spread occurs, and there is asymptomatic or mild febrile undifferentiated illness, such as fever, malaise, headache, nausea, gastrointestinal disturbances, and sore throat, or combination of these.

Illustration of a Poliovirus

Reference: http://www.brown.edu/Courses/Bio_160/Projects1999/polio/images/polio1.jpg

Diagram of an African Green Monkey Cell

Reference : http://www.nature.com/

2) Plants
Plants are used in the study of Tobacco mosaic virus and this is based on the virus plaques on the leaves of plants to determine the virus numbers.

A photomicrograph of Tobacco Mosaic Virus (TMV)

Reference : http://www.erec.ifas.ufl.edu/
The Tobacco mosaic virus (TMV) is an RNA virus that infects plants, tobacco in particular, showing characteristic patterns like mottling and discoloration on the leaves, hence the name. It was the first virus to be discovered.

Picture of a leaf used in the study of TMV (Gradual changes shown in A and B)

Reference : http://www.apsnet.org/
Fig. 1. Tobacco mosaic virus (TMV). (A) Systemic infections of Nicotiana tabacum cv. Turk plants showing TMV-associated mosaic. (B) Necrotic local lesions on N. tabacum Glurk leaf, demonstrating Holmes’ N-gene resistance following inoculation with TMV.

3) Tissue Culture

In cell tissue culture, the cells are grown in vitro, which means they are grown in test tubes.

Primary cell cultures typically will have a finite life span in culture whereas continuous cell lines are, by definition, abnormal and are often transformed cell lines.

a) Primary Cell Cultures

b) Continuous Cell Lines

They are derived from primary cell lines and are transformed/cancerous cells. They can exist in either polyploid form or multiploid form and theoretically can be sub-cultured indefinitely. It is a method of choice of cultivating viruses, where possible.

Photo micrograph of continuous cell lines (cancerous cells present)

Reference : http://research.nki.nl/agamilab/images/p27.jpg