Article on nutritional requirements,cultivation and isolation of viruses
Answers
*Isolation of Viruses:
Viruses are isolated from infected host cells containing mature virions. The cells are mechanically disrupted and the cell contents are released in a suitable buffer solution. The suspension containing the virions and cell ingredients is then subjected to centrifugation for several times at different speeds to fractionate the virions from other cell components. Such procedure is known as differential centrifugation.
A more refined technique is the density gradient centrifugation which is usually applied for getting a more purified sample of viruses. Before subjecting the sample of virus to density gradient centrifugation, it is initially purified by differential centrifugation. A density gradient is prepared in a centrifuge tube. For example, a sucrose gradient contains a linearly increasing concentration of sucrose from top to bottom of the centrifuge tube.
The partially purified virus sample is poured on the top and the tube is subjected to high-speed centrifugation for several hours in an ultracentrifuge. The centrifugal force drives the viral particles towards the bottom until they settle at a density gradient of sucrose which equals that of the virions, forming a concentrated zone or band.
The suspension of virions can then be removed from this band with the help of a pipette. The bacteriophages causing lytic infections can be isolated by a more or less similar method by differential centrifugation of the lysate to eliminate cell debris and the non-lysed intact cells of the host bacteria.
*Cultivation of Viruses:
Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (Figure 6.3.2a ). The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected (Figure 6.3.1b ). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.
Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.
The embryo or host animal serves as an incubator for viral replication (Figure 6.3.3 ). Location within the embryo or host animal is important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.
