6.1. Virus Isolation

Viruses are obligate intracellular parasites that require living cells in order to replicate. Generally cell culture, embryonated eggs and small laboratory animals are used for the isolation of viruses. Embryonated eggs are very useful for the isolation of influenza and paramyxoviruses. Although laboratory animals are useful in isolating different kind of viruses, cell culture is still a preferred way for virus isolation in many of the laboratories.

For primary cell cultures, tissue fragments are first dissociated into small pieces with the help of scissors and addition of trypsin. The cell suspension is then washed couple of times with minimal essential media and seeded into a flat-bottomed glass or plastic container bottle after resuspending it with a suitable liquid medium and fetal calf serum. The cells are kept in incubator at 37°C for 24 to 48hrs depending on the cell type. This allows the cells to attach the surface of the container and its division following the normal cell cycle.

Cell cultures are generally of 3 types:-

1. 1. Primary culture – These are prepared directly from animal or human tissues and can be subcultured only once or twice e.g. chicken embryo fibroblast.

2. Diploid cell culture – They are derived from neonatal tissues and can be subcultured 5-10 times. e.g. human diploid fibroblasts cells.

3. Continuous cells – They are derived from tumor tissues and can be subcultured more than 10 times. e.g. Vero, Hep2, Hela.

Specimens containing virus should be transported to the laboratory as soon as possible upon being taken. Oral or cloacal swabs should be collected in vials containing virus transport medium. Body fluids and tissues should be collected in a sterile container and sealed properly. If possible all the samples should be maintained and transported in a cold condition for higher recovery rates.

Upon receipt, the samples should be inoculated into cell culture depending on the history and symptoms of the disease. The infected cell culture flask should be observed every day for any presence of cytopathic effect (CPE). Certain kind of samples, such as faeces and urine are toxic to the cell cultures and may produce a CPE-like effect. When virus specific CPE is evident, it is advised to passage the infected culture fluid into a fresh cell culture. For cell-associated viruses such as cytomegaloviruses, it is required to trypsinize and passage the intact infected cells. Viruses such as adenovirus can be subcultured after couple of time freezing and thawing of the infected cells.