Principles of Virology. Jane Flint
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cells which contact the basement membrane. In the alveoli the epithelium is made of a thin, single cell layer to facilitate air exchange. Air-liquid interface cultures may be produced from primary human bronchial cells or respiratory cell lines (Fig. 2.4).
Because viruses are obligatory intracellular parasites, they cannot reproduce outside a living cell. An exception comes from the demonstration in 1991 that infectious poliovirus could be produced in an extract of human cells incubated with viral RNA, a feat that has not been achieved for any other virus. Consequently, most analyses of viral replication have used cultured cells, embryonated eggs, or laboratory animals. For a discussion of whether to call these different systems in vivo or in vitro, see Box 2.4.
Evidence of Viral Reproduction in Cultured Cells
Before quantitative methods for measuring viruses were developed, evidence of viral propagation was obtained by visual inspection of infected cells. Some viruses kill the cells in which they reproduce, and they may eventually detach from the cell culture plate. As more cells are infected, the changes become visible and are called cytopathic effects.
Many types of cytopathic effect can be seen with a simple light or phase-contrast microscope at low power, without fixing or staining the cells. These changes include the rounding up and detachment of cells from the culture dish, cell lysis, swelling of nuclei, and sometimes the formation of a group of fused cells called a syncytium (Fig. 2.5). High-power microscopy is required for the observation of other cytopathic effects, such as the development of intracellular masses of virus particles or unassembled viral components in the nucleus and/or cytoplasm (inclusion bodies), formation of crystalline arrays of viral proteins, membrane blebbing, duplication of membranes, and fragmentation of organelles.
Figure 2.4 Production of airway-liquid interface cultures of bronchial epithelium. (A) Epithelial cells are seeded onto a permeable membrane and cell culture medium is supplied on both apical (top) and basal (bottom) sides. (B) When the cells are confluent, medium on the apical side is removed. Contact of the cells with air drives differentiation of cells towards types found in the airways, such as goblet cells, ciliated and nonciliated cells, and basal cells. Cultures may be produced that mimic tracheobronchial cells, with different cell types, or human alveolar cells with only two cell types (not shown).
TERMINOLOGY
In vitro and in vivo
The terms “in vitro” and “in vivo” are common in the virology literature. In vitro means “in glass” and refers to experiments carried out in an artificial environment, such as a glass or plastic test tube. Unfortunately, the phrase “experiments performed in vitro” is used to designate not only work done in the cell-free environment of a test tube but also work done within cultured cells. The use of the phrase in vitro to describe living cultured cells leads to confusion and is inappropriate. In vivo means “in a living organism” but may be used to refer to either cells or animals. Those who work on plants avoid this confusion by using the term “in planta.”
In this textbook, we use in vitro to designate experiments carried out in the absence of cells, e.g., in vitro translation. Work done in cells in culture is done ex vivo, while research done in animals is carried out in vivo.
The time required for the development of cytopathology varies considerably among animal viruses. For example, depending on the size of the inoculum, enteroviruses and herpes simplex virus can cause cytopathic effects in 1 to 2 days and destroy the cell monolayer in 3. In contrast, cytomegalovirus, rubella virus, and some adenoviruses may not produce such effects for several weeks.
Figure 2.5 Development of cytopathic effect. (A) Cell rounding and lysis during poliovirus infection. Shown are uninfected cells (upper left) and cells 5.5 h after infection (upper right), 8 h after infection (lower left), and 24 h after infection (lower right). (B) Syncytium formation induced by murine leukemia virus. The field shows a mixture of individual refractile small cells and flattened syncytia (arrow), which are large, multinucleated cells. Courtesy of R. Compans, Emory University School of Medicine. (C) Schematic illustration of syncytium formation. Viral glycoproteins on the surface of an infected cell bind receptors on a neighboring cell, causing fusion.
The development of characteristic cytopathic effects in infected cell cultures is frequently monitored in diagnostic virology after isolation of viruses from specimens obtained from infected patients or animals. In the research laboratory, observation of cytopathic effect can be used to monitor the progress of an infection, and is often one of the phenotypic traits that characterize mutant viruses.
Some viruses multiply in cells without causing obvious cytopathic effects. For example, many members of the families Arenaviridae, Paramyxoviridae, and Retroviridae do not cause obvious damage to cultured cells. Infection by such viruses must therefore be assessed using alternative methods, as described in “Assay of Viruses” below.
Embryonated Eggs
Before the advent of cell culture, many viruses were propagated in embryonated chicken eggs (Fig. 2.6). At 5 to 14 days after fertilization, a hole is drilled in the shell and virus is injected into the site appropriate for its replication. This method of virus propagation is now routine only for influenza virus. The robust yield of this virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production.
Laboratory Animals
In the early 1900s, when viruses were first isolated, freezers and cell cultures were not available, and it was necessary to maintain virus stocks by continuous passage from animal to animal. This practice not only was inconvenient but also, as we shall see, led to the selection of viral mutants (Volume II, Chapter 7). For example, monkey-to-monkey intracerebral passage of poliovirus selected a mutant that could no longer infect chimpanzees by the oral route, the natural means of infection.
Although cell culture has supplanted animals for propagating most viruses, experimental infection of laboratory animals has always been, and will continue to be, obligatory for studying the processes by which viruses cause disease. The study in monkeys of poliomyelitis, the paralytic disease caused by poliovirus, led to an understanding of the basis of this disease and was instrumental in the development of a successful vaccine. Similarly, the development of vaccines against hepatitis B virus would not have been possible without experimental studies with chimpanzees. Understanding how the immune system or any complex organ reacts to a virus cannot be achieved without research on living animals. The development of viral vaccines, antiviral drugs, and diagnostic tests for veterinary medicine has also benefited from research on diseases in laboratory animals. Despite their utility, it must be appreciated