Principles of Virology. Jane Flint
Studies of the interactions between viruses and the immune system are particularly instructive, because of the many viral countermeasures that can frustrate this system. Elucidation of these measures continues to teach us about the basis of immunity (Volume II, Chapters 2 to 4).
Cultivation of Viruses
Cell Culture
Types of Cell Culture
Although human and other animal cells were first cultured in the early 1900s, contamination with bacteria, mycoplasmas, and fungi initially made routine work with such cultures extremely difficult. For this reason, most viruses were produced in laboratory animals. The use of antibiotics in the 1940s to control microbial infection was crucial to the establishment of the first cell lines, such as mouse L929 cells (1948) and HeLa cells (1951). John Enders, Thomas Weller, and Frederick Robbins discovered in 1949 that poliovirus could multiply in cultured cells. As noted in Chapter 1, this revolutionary finding, for which these three investigators were awarded a Nobel Prize in 1954, led the way to the propagation of many other viruses in cells in culture, the discovery of new viruses, and the development of vaccines such as those against the viruses that cause poliomyelitis, measles, and rubella. The ability to infect cultured cells synchronously permitted studies of the biochemistry and molecular biology of viral reproduction. Large-scale propagation and purification of virus particles allowed studies of the composition of virus particles, leading to the solution of high-resolution, three-dimensional structures (see Chapter 4).
Cells in culture are still the most commonly utilized hosts for the propagation of animal viruses. To prepare a cell culture, tissues are dissociated into a single-cell suspension by mechanical disruption followed by treatment with proteolytic enzymes. The cells are then suspended in culture medium and placed in specialized plastic flasks or covered plates. As the cells divide, they cover the plastic surface. Epithelial and fibroblastic cells attach to the plastic and form a monolayer, whereas blood cells such as lymphocytes settle but do not adhere. The cells are grown in a chemically defined and buffered medium optimal for their growth. Commonly used cell lines double in number in 24 to 48 h in such media. Most cells retain viability after being frozen at low temperatures (−70 to −196°C).
There are three main kinds of monolayer cell cultures (Fig. 2.2), each with advantages and disadvantages for virus research. Primary cell cultures are prepared from animal tissues as described above. They have a limited life span, usually no more than 5 to 20 cell divisions. Commonly used primary cell cultures are derived from chicken or mouse embryos, monkey kidneys, or human tissues that are otherwise typically disposed of, such as embryonic amnion, kidney, foreskin, and respiratory epithelium. Such cells are used for experimental virology when the state of cell differentiation is important or when appropriate cell lines are not available. They are also used in vaccine production: for example, infectious attenuated poliovirus vaccine strains may be propagated in primary monkey kidney cells. Primary cell cultures are used for the propagation of viruses to be used as human vaccines to avoid contamination of the product with potentially oncogenic DNA from continuous cell lines (see below). Some viral vaccines are now prepared in diploid cell strains, which consist of a homogeneous population of a single cell type and can divide up to 100 times before dying. Despite the numerous divisions, these cells retain the diploid chromosome number. The most widely used diploid cells are those established from human embryos, such as the WI-38 strain derived from human embryonic lung.
Figure 2.2 Different types of cell culture used in virology. Confluent cell monolayers photographed by low-power light microscopy. (A) Primary human foreskin fibroblasts; (B) established line of mouse fibroblasts (3T3); (C) continuous line of human epithelial cells (HeLa [Box 2.3]). The ability of transformed HeLa cells to overgrow one another is the result of a loss of contact inhibition. Courtesy of R. Gonzalez, Princeton University.
Continuous cell lines consist of a single cell type that can be propagated indefinitely in culture. These immortal lines are usually derived from tumor tissue or by treating a primary cell culture or a diploid strain with a mutagenic chemical or an oncogene. Such cell lines often do not resemble the cell of origin; they are less differentiated (having lost the morphology and biochemical features that they possessed in the organ), are often abnormal in chromosome morphology and number (aneuploid), and can be tumorigenic (i.e., they produce tumors when inoculated into immunodeficient mice). Examples of commonly used continuous cell lines include those derived from human carcinomas (e.g., HeLa [Henrietta Lacks] cells [Box 2.2]) and from mice (e.g., L and 3T3 cells). Continuous cell lines provide a uniform population of cells that can be infected synchronously for growth curve analysis (see “The One-Step Growth Cycle” below) or biochemical studies of virus replication.
In contrast to cells that grow in monolayers on plastic dishes, others can be maintained in suspension cultures, in which a spinning magnet continuously stirs the cells. The advantage of suspension culture is that a large number of cells can be grown in a relatively small volume. This culture method is well suited for applications that require large quantities of virus particles, such as X-ray crystallography or production of vectors.
Despite the wide utility of monolayer and suspension cell cultures in virology, they are not without limitations, including the finite life span of primary cell cultures and the abnormal phenotype of continuous cell lines, such as immortality. These problems can be overcome by the use of induced pluripotent stem cells (iPSCs), which are adult cells that have been reprogrammed genetically to an embryonic stem-cell like state by the introduction of four genes (Oct4, Sox2, Kif4, and cMyc). They are most commonly made from human fibroblasts, although other cell types have been used. Such iPSCs can be differentiated into many different cell types, such as cardiomyocytes, neurons, and hepatocytes, by treatment with specific growth factors. Viral reproduction can be studied in specific human cell types using cells derived from iPSCs.
BACKGROUND
The cells of Henrietta Lacks
The most widely used continuous cell line in virology, the HeLa cell line, was derived from Henrietta Lacks. In 1951, the 31-year-old mother of five visited a physician at Johns Hopkins Hospital in Baltimore and was found to have a malignant tumor of the cervix. A sample of the tumor was taken and given to George Gey, head of tissue culture research at Hopkins. Gey had been attempting for years, without success, to produce a line of human cells that would live indefinitely. When placed in culture, Henrietta Lacks’ cells propagated as no other cells had before.
On the day in October that Henrietta Lacks died, Gey appeared on national television with a vial of her cells, which he called HeLa cells. He said, “It is possible that, from a fundamental study such as this, we will be able to learn a way by which cancer can be completely wiped out.” Soon after, HeLa cells were used to propagate poliovirus, which was causing poliomyelitis throughout the world, and they played an important role in the development of poliovirus vaccines. Henrietta Lacks’ HeLa cells started a medical revolution: not only was it possible to propagate many different viruses in these cells, but the work set a precedent for producing continuous cell lines from many human tissues.