2.4
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 that all animal models are surrogates for the events that occur during viral infections of humans.
Assay of Viruses
There are two main types of assay for detecting viruses: biological and physical. Because viruses were first recognized by their infectivity, the earliest assays focused on this most sensitive and informative property. However, biological assays such as the plaque assay and end-point titration methods do not detect noninfectious particles. In contrast, all particles are accounted for with physical assays such as electron microscopy or by immunological methods. Knowledge of the number of noninfectious particles is useful for assessing the quality of a virus preparation.
Measurement of Infectious Units
One of the most important procedures in virology is measuring the virus titer, the concentration of infectious virus particles in a sample. This parameter is determined by inoculating serial dilutions of virus into host cell cultures, chicken embryos, or laboratory animals and monitoring for evidence of virus multiplication. The response may be quantitative (as in assays for plaques, fluorescent foci, infectious centers, or abnormal growth and morphology) or all-or-none, in which the presence or absence of infection is measured (as in an end-point dilution assay). Please note that “titer” is not a verb.
Figure 2.6 Growth of viruses in embryonated eggs. The cutaway view of an embryonated chicken egg shows the different routes by which viruses are inoculated into eggs and the distinct compartments in which particular viruses may propagate.
Figure 2.7 Plaques formed by different animal viruses. (A) Photomicrograph of a single plaque formed by pseudorabies virus in bovine kidney cells. Shown are unstained cells (left) and cells stained with the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), which is converted to a blue compound by the product of the lacZ gene carried by the virus (right). Courtesy of B. Banfield, Princeton University. (B) Plaques formed by poliovirus on human HeLa cells stained with crystal violet. (C) Illustration of the sequential spread of a cytopathic virus from an initial infected cell to neighboring cells, resulting in a plaque.
Plaque Assay
