In the previous chapter, viruses were defined as obligate intracellular parasites that are unable to direct any independent biosynthetic processes outside the host cell. It was further noted that the genetic complexity of viruses varies greatly between individual virus families, ranging from those viruses that encode only a few proteins to others that encode several hundred proteins.
Given this remarkable diversity, it is hardly surprising that the replication processes used by individual viruses would also be highly variable. However, all viruses must progress through the same general steps for replication to occur. Specifically, all viruses must attach to a susceptible host cell, enter the cell, disassemble the virus particle (uncoating), replicate its own genetic material and express the associated proteins, assemble new virus particles, and escape from the infected cell (release). This chapter will outline the general processes involved in each of these steps.
GROWTH OF VIRUSES
Before the development of in vitro cell culture techniques, viruses had to be propagated in their natural host. For bacterial viruses (bacteriophages), this was a relatively simple process. Consequently, scientists were able to develop laboratory-based research methods to study bacteriophages long before they were able to conduct comparable studies with plant or animal viruses. For animal viruses, samples from affected animals were collected and used to infect other animals, initially of the same species.
When consistent results were obtained, attempts were usually made to determine whether other species might also be susceptible. These types of experiments were performed in an effort to determine the host range of any presumed viral agent. Although progress was made in defining the biological properties of viruses, this manner of propagation had obvious major drawbacks, especially with viruses affecting large animals.
A most serious issue was the infection status of the recipient animals. For example, an undetected infectious agent in a sheep could alter the clinical signs observed after inoculation of that sheep with the test agent, and samples collected from this individual might now include several infectious agents, potentially confounding future experiments.
In an attempt to avoid this type of contamination problem, animals that were to be used in research studies were raised under more defined conditions. As new infectious agents were discovered and tests developed for their detection, the research animals became more “clean” and the concept of the “specific pathogenfree” (SPF) animal was born. It is noteworthy; however, that animals that were thought to be specific pathogen free could be infected with pathogens that were still undefined or undeclared.
For example, pneumonia virus of mice (mouse pneumovirus) was discovered when “uninfected” control animals inoculated with lung extracts from other control animals died during experimental influenza virus infection studies. Many early virological and immunological studies were compromised by using rodents unknowingly infected with mouse hepatitis virus, lactate dehydrogenase-elevating virus, or other agents. Although live animals are no longer commonly used for routine virus isolation/propagation, animals are used still extensively for assessing viral properties such as virulence, pathogenesis, and immunogenicity.
The search for culture systems suitable for the propagation and study of viruses led to the discovery, in 1931, that vaccinia virus and herpes simplex virus could be grown on the chorioallantoic membrane of embryonated chicken eggs, as was already known for fowlpox virus, a pathogen of birds. It was soon determined that viruses in many families of animal viruses can be grown in embryonated eggs, probably because of the wide variety of cell and tissue types present in the developing embryo and its environment. Consequently, the embryonated chicken egg became a standard culturing system for routine isolation and propagation of avian viruses and select mammalian viruses. In some cases, embryonated eggs entirely replaced research animals for the growth of virus stocks, and if the viral infection resulted in the death of the embryo, this system could also be used to quantify (titrate) the amount of virus in a virus stock or specimen (as described in greater detail later in this chapter). The egg system, which is labor-intensive and expensive, has largely been replaced by vertebrate cell culture-based systems; however, it is still widely used for the isolation and growth of influenza viruses and many avian viruses. Various in vitro cell culture systems have been utilized since artificial medium was developed to maintain cell viability outside the source animal. These include organ cultures, explant cultures, primary cell cultures, and cell lines. An organ culture consists of an intact organ, which maintains the cellular diversity and the three-dimensional structure of the tissue. Organ cultures are utilized for short-term experiments. Explant cultures consist of portions (eg, a slice or fragment) of an organ or tissue. Although explant cultures lack the complexity of the intact organ, their cellular components exist in a state that more closely models the in vivo environment than do cells propagated as primary cell cultures or cell lines. The creation of primary cell cultures utilizes proteases such as trypsin or collagenase to disassociate individual cells of a given tissue such as fetal kidney or lung. The individual cells are then permitted to attach to a cell culture matrix on which they will divide for a limited number of cell divisions. The limited lifespan of most primary cells requires continual production of the cells from new tissue sources, which can lead to variable cell quality between batches. This problem was largely overcome with the generation of immortalized cell lines, which in theory are capable of unlimited cell divisions. Initially, the generation of immortalized cell lines (transformation) was an empirical process with a low probability of success but it is now possible to immortalize virtually any cell type, so the number of cell lines representing different species is increasing rapidly. The advent of in vitro animal cell culture brought research studies on animal viruses in line with those involving bacteriophage, and enhanced the quality and reliability of diagnostic testing. The ability to isolate and propagate animal viruses in cultured cells also made it possible to identify viruses as the etiologic agents of specific diseases through the successful application of Koch’s postulates. Fulfilment of Koch’s postulates requires that the infectious agent be isolated in pure culture; an achievement that was not possible for viruses prior to the development of cell culture systems. Replacement of living animals with cell culture systems decreased, but did not entirely eliminate, the problems associated with the presence of adventitious viruses. For example, early batches of the modified live poliovirus vaccine were contaminated with SV40 virus, a simian polyomavirus originating from the primary monkey kidney cultures used for vaccine production. Similarly, interpretation of the results of some early studies on newly described parainfluenza viruses is complicated because of virus contamination of the cell cultures used for virus isolation. Contamination of ruminant cell cultures with bovine viral diarrhea virus has been an especially insidious and widespread problem. Some contaminated cell cultures and lines were probably derived from infected fetal bovine tissue, but far more commonly, cells became infected through exposure to fetal bovine serum contaminated with bovine viral diarrhea virus. Fetal bovine serum became a standard supplement for cell culture medium in the early 1970s. The fact that many ruminant cell lines became infected from contaminated serum has compromised much research pertaining to ruminant virology and immunology, confounded diagnostic testing for bovine viral diarrhea virus and caused substantial economic losses as a result of contaminated vaccines. The extent of the problem was not fully defined until the late 1980s when high quality diagnostic reagents became available. As with experimental animals, problems with contaminating viral infections of cell cultures were only defined when the existence of the relevant infectious agent became known. Standard protocols for the use of serum in biological production systems now require irradiation of the serum to inactivate all viruses, known or unknown. With current technology allowing amplification and detection of virtually all nucleic acid species in cells, coupled with rapid sequencing of these products, a complete profile of cell cultures for contaminating organisms is now feasible.
Recognition of Viral Growth in Culture
Prior to the development of cell culture systems, identifying the presence of a viral agent in a plant or animal host was dependent upon the recognition of signs not found in an unaffected (control) host, death being the most extreme outcome and easiest to determine. Similarly, the presence of a replicating virus in cultured cells can be detected by identifying specific cellular characteristics that arise as a consequence of virus infection. In broad terms, any observable cellular characteristic that is present in virusinfected cells and which is absent in uninfected cells maintained under identical growth conditions is referred to as a cytopathic effect (CPE). Virus-induced forms of CPE are generally observed through microscopic examination of the test culture system (Fig. 2.1). The most common forms of CPE observed in cultured cells are cell lysis and significant changes in cell morphology. Examples of morphological changes include the rounding, clumping, shrinkage, and detachment of individual cells from the cell culture matrix. Virus-induced fusion of neighboring cells represents another form of CPE. For example, cells infected with avian reovirus commonly fuse to form multinucleated cells or syncytia (Fig. 2.1A). Many members of the family Paramyxoviridae can cause this type of morphological change in cultured cells, but the extent of syncytium formation is cell type dependent. The type of cytopathology noted in culture can be characteristic for a given class of virus. For example, alphaherpesviruses produce distinct cytopathology characterized by rounded cells, with or without small syncytia, which spreads very rapidly through a susceptible cell culture (Fig. 2.1B). Cells infected with some types of viruses acquire the ability to bind (adsorb) red blood cells (syn., erythrocytes) on their surface; a property referred to as hemadsorption. For example, cells infected with bovine parainfluenza virus 3 adsorb chicken red blood cells to the plasma membrane (Fig. 2.1D). Binding of red blood cells to the surface of the infected cell is actually mediated by viral glycoproteins that are expressed on the cell surface and which bind to receptors on the red blood cells. Consequently, hemadsorption only occurs with viruses that bud from the plasma membrane, and may be specific for red blood cells of a given animal species. Viruses that induce hemadsorption also show the ability to hemagglutinate red blood cells in cell-free medium. As discussed later in the chapter, this property can be used as the basis for quantifying the amount of virus within a sample. The same viral proteins that permit hemadsorption are also responsible for the hemagglutination reaction. There are, however, viruses that can themselves hemagglutinate red blood cells but not cause hemadsorption to cells infected with the same virus (eg, adenoviruses and alphaviruses). Another type of morphological change commonly observed in virus-infected cells is the formation of inclusion bodies (Fig. 2.2). Inclusion bodies are intracellular abnormalities, commonly new structures, which arise as a direct consequence of virus infection. Inclusion bodies can be observed with a light microscope after fixation and treatment with cytological stains, but, as with hemadsorption, not all viruses will produce obvious inclusion bodies. The type of virus infecting a cell can be inferred by the location and shape of the inclusions. For example, cells infected with herpesviruses, adenoviruses, and parvoviruses can have intranuclear inclusions, whereas cytoplasmic inclusions are characteristic of infections with poxviruses, orbiviruses, and paramyxoviruses (Fig. 2.2B,C). The composition of the inclusions will vary with the virus type. The cytoplasmic Negri bodies identified in rabies virus-infected cells are composed of aggregates of nucleocapsids, whereas the intranuclear inclusions that occur in adenovirus-infected cells consist of crystalline arrays of mature virus particles (Fig. 2.2D). Cytological stains are rarely used to identify cells infected with specific viruses, but are mainly used as a screening test to assess the presence of any virus. In the absence of a metagenomic screening procedures, detection of viruses that produce no cytopathology (CPE), do not induce hemadsorption or hemagglutinate, or produce no definable inclusions, is accomplished using virus-specific tests. For example, this is the case in screening bovine cells for the presence of noncytopathic bovine viral diarrhea virus. The most commonly used tests in this type of situation are immunologically based assays such as the fluorescent antibody assay (immunofluorescence assay, IFA) or immunohistochemical staining assay (Fig. 2.3). The quality of these assays is dependent on the specificity of the antibodies that are used. With the development of monoclonal antibodies and monospecific antisera, this issue has been largely resolved. Other virus-specific tests are based on the detection of virusspecific nucleic acid in the infected cells. Initially, assays of this sort relied on the use of nucleic acid probes capable of hybridizing in a sequence-specific manner with the target nucleic acid. Hybridizationbased assays have largely been replaced by those based on polymerase chain reaction (PCR) because of their enhanced sensitivity and ease of performance (see Chapter 5: Laboratory Diagnosis of Viral Infections).
'Fenner's Veterinary Virology (5판)' 카테고리의 다른 글
| [Chapter 1] The Nature of Viruses (0) | 2025.03.08 |
|---|