Viral Culture techniques
The laboratory culture of viruses is more complex than that required for most other microorganisms. Unlike bacteria and fungi, most of which can be grown on relatively simple media (such as blood agar, MacConkey agar or Sabouraud's agar), viruses will only replicate inside living cells because viruses are obligate intracellular parasites. Cell cultures are much less cumbersome than inoculations of laboratory animals or embryonated eggs and are employed for viral growth whenever possible.
Cell culture and 'cell lines'
Cells derived from mammalian tissues will form as a thin sheet (a "monolayer") on the surface of suitable glass or plastic containers. They are supplied with nutrients by special sterile cell culture liquid media, which must always completely cover the cell sheet or the cells will die.
Normal cells can be subcultured a finite number of times, after which they will no longer grow in culture (human fibroblasts survive 40-50 subcultures). Cancer cells or normal cells which have been ‘transformed’ by chemicals or viruses can be subcultured indefinitely and are said to have been immortalised to become cell lines. Some of these cell lines, such as HeLa cells, have been growing in laboratories throughout the world for over half a century.
Many different cell lines are available, but only a fraction of these are utilised in virology laboratories. Cell lines retain some of the characteristics of the tissue from which they were originally derived. There are two main types of lines used in virology laboratories.
- Those derived from ectodermal cells (e.g. skin, mucous membranes and carcinomas) are known as 'epithelioid' cell lines;
- those derived from mesodermal cells (e.g. connective tissue and sarcomas) are known as 'fibroblastic' cell lines.
The two types of cells differ markedly in appearance: epithelioid cells are polygonal or cuboidal in shape and have a ‘cobblestone’ appearance; while fibroblastic cells are thin and elongated in shape, and appear as swirls of 'spindle-shaped' cells.
Epithelioid cells and Fibroblastic cells
Some viruses are fastidious and will only grow in certain types of cells, either epitheloid or fibroblastic, so several different cell lines will be inoculated when attempting to isolate an unknown virus from clinical specimens. In addition some lines have been derived from other cell types such as lymphocytes – the latter are essential for the laboratory isolation of strictly lymphotropic viruses such as the human immunodeficiency virus (HIV).
Virus‑induced CPEs in cell cultures
Most viruses will successfully replicate in routine cell cultures. When cultivable viruses are inoculated into a suitable cell culture the viral particles will firstly attach to the cell membranes, then penetrate and multiply inside the cells. Virus particles are then released into the culture medium. This process may be very short (6-12 hours) or can take up to 5-7 days, depending on the type of virus.
Many viruses kill or damage the cells in which they have replicated: this results in a visible abnormality which may be detected via microscopy and is referred to as the "cytopathic effect" (CPE) of the particular virus. Certain viruses produce a characteristic CPE in cell culture, and this often assists in recognising the growth of that virus in the laboratory.
For example:
| CPE Observe | Possible Virus |
|---|---|
| Uniform rounding and ballooning of cells | Herpes simplex virus |
| Focal rounding, loss of cells | Cytomegalovirus |
| Uniform shrinking, rounding, loss of cells | Enteroviruses e.g. polio |
| Grape-like clusters of cells | Adenoviruses |
| None or minimal, occasional syncytial or ‘giant cell’ formation | Myxoviruses and paramyxoviruses such as influenza, measles and rubella |
Virus neutralisation
Specific antibodies raised against viral attachment proteins prevent the entry of the virus into the cell. This is called 'neutralisation' of the virus and is an exquisitely sensitive and highly specific reaction.
Apart from its importance in conferring immunity, neutralisation is also an important way of identifying viruses in the laboratory.