Since culturing viruses requires a living host, early investigators had trouble propagating adequate quantities of the specific viruses they wished to study. Virtually all of the initial attempts at cultivation had to be performed in an organism that was the usual host for the virus. But this method had its limitations. How could researchers have ever traced the stages of viral multiplication if they had been restricted to the natural host, especially in the case of human viruses? Fortunately, systems of cultivation with broader applications were developed, including in vitro cell (or tissue) culture methods and in vivo inoculation of laboratory-bred animals and embryonic bird tissues. Such use of substitute host systems permits greater control, uniformity, and wide-scale harvesting of viruses.
The primary purposes of viral cultivation are (1) to isolate and identify viruses in clinical specimens; (2) to prepare viruses for vaccines; and (3) to do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells.
Using Cell (Tissue) Culture Techniques
The most important early discovery that led to easier cultivation of vi ruses in the laboratory was the development of a simple and effective way to grow populations of isolated animal cells in culture. These types of in vitro cultivation systems are termed cell culture or tissue culture. This method makes it possible to propagate most viruses. Much of the virologist’s work involves developing and maintaining these cultures. Animal cell cultures are grown in sterile chambers with special media that contain the correct nutrients required by animal cells to survive. The cultured cells grow in the form of a monolayer, a single, confluent sheet of cells that supports viral multiplication and permits close inspection of the culture for signs of infection (figure 1).
Fig1. Microscopic views of normal and infected cell cultures. (a) Cell culture plate infected with herpes simplex virus. Clear, round spaces are regions of infection (plaques) where the host cells have been destroyed by viruses. (b) Close-up of a normal, uninfected region of cultured cells. (c) Close-up of plaques, which consist of open spaces where cells have been disrupted by viral infection. (a): Marylou Gibson, PhD, VIRAPUR; (b and c): CDC
Cultures of animal cells usually exist in the primary or continuous form. Primary cell cultures are prepared by placing freshly isolated ani mal tissue in a growth medium. Embryonic, fetal, adult, and even can cerous tissues have served as sources of primary cultures. A primary culture retains several characteristics of the original tissue from which it was derived, but this original line generally has a limited existence. Eventually it will die out or mutate into a line of cells that can grow by continuous subculture in fresh nutrient medium. One very clear advantage of cell culture is that a specific cell line can be available for viruses with a very narrow host range. Strictly human viruses can be propagated in one of several primary or continuous human cell lines, such as embryonic kidney cells, fibroblasts, bone marrow, or heart cells.
One way to detect the growth of a virus in culture is to observe degeneration and lysis of infected cells in a monolayer of a cell culture (figure 1a). The areas where virus-infected cells have been destroyed show up as clear, well-defined patches in the cell sheet called plaques (figure 1c). Plaques are essentially the macroscopic manifestation of cytopathic effects (CPEs). This same technique is used to detect and count bacteriophages, because they also produce plaques when grown in soft agar cultures of their host cells. A plaque develops when the viruses released by an infected host cell radiate out to adjacent host cells. As new cells become infected, they die and release more viruses, and so on. As this process continues, the infection spreads gradually and symmetrically from the original point of infection, causing the macroscopic appearance of round, clear spaces that correspond to areas of lysed cells.
Using Bird Embryos
An embryo is an early developmental stage of animals marked by rapid differentiation of cells. Birds undergo their embryonic period within the closed protective case of an egg, which makes an incubating bird egg a nearly perfect system for viral propagation. It is an intact and self-supporting unit, complete with its own sterile environment and nourishment. Furthermore, it furnishes several embryonic tissues that readily support viral multiplication. Every year, hundreds of millions of chicken embryos are inoculated to prepare influenza vaccines.
Chicken, duck, and turkey eggs are the most common choices for inoculation. The egg must be injected through the shell, necessitating rigorous sterile techniques to prevent contamination by bacteria and fungi from the air and the outer surface of the shell. The exact tissue inoculated is guided by the type of virus being cultivated and the goals of the experiment (figure 2).
Fig2. Cultivating animal viruses in a developing bird embryo. (a) A technician inoculates fertilized chicken eggs with influenza virus. (b) The shell is perforated using sterile techniques, and a virus preparation is injected into a site selected to grow the viruses. Targets include the allantoic cavity, a sac for embryonic waste removal; the amniotic cavity that cushions and protects the embryo; the chorioallantoic membrane, for embryonic gas exchange; the yolk sac, for the nourishment of the embryo; and the embryo itself. (a): Greg Knobloch/CDC
Viruses multiplying in embryos may or may not cause effects visible to the naked eye. The signs of viral growth include death of the embryo, defects in embryonic development, and localized areas of damage in the membranes, resulting in discrete, opaque spots called pocks (a variant of pox). Embryonic fluids and tissues can be prepared for direct examination with an electron microscope. Certain viruses can also be detected by their ability to agglutinate red blood cells (form big clumps) or by their reaction with an antibody of known specificity that will affix to its corresponding virus, if it is present.
Using Live Animal Inoculation
Specially bred strains of white mice, rats, hamsters, guinea pigs, and rabbits are the usual choices for animal cultivation of viruses. Invertebrates or nonhuman primates are occasionally used as well. Because viruses can exhibit host specificity, certain animals can propagate a given virus more readily than others. Depending on the particular experiment, tests can be performed on adult, juvenile, or newborn animals. The animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood, muscle, body cavity, skin, or footpads.
