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).
FIGURE 2.1 Cytopathic effects produced by different viruses. The cell monolayers are shown as they would normally be viewed by phase contrast microscopy, unfixed and unstained. (A) Avian reovirus in Vero cells with prominent syncytium (arrow). (B) Untyped herpesvirus in feline lung cell. (C) Bovine viral diarrhea virus in primary bovine kidney cells. (D) Parainfluenza virus 3 in Vero cells detected by hemadsorption of chicken red blood cells. Courtesy of E. Dubovi, Cornell University.
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).
VIRUS REPLICATION
A fundamental characteristic that separates viruses from other replicating entities is the manner in which new virus particles are synthesized. Unlike eukaryotic and prokaryotic cells, which increase their numbers through the processes of mitosis and binary fission, respectively, new virus particles are assembled de novo from the various structural components that are synthesized during the virus infection. The earliest recognition of this unique replication pattern came from studies using bacteriophage.
The outline of the experimental proof of concept was relatively simple: (1) add a chloroform-resistant phage to a culture of bacteria for several minutes; (2) rinse the bacteria to remove nonattached phage; (3) incubate the culture and remove samples at various periods of time; (4) treat sampled bacterial cultures with chloroform to stop growth; (5) quantify the amount of phage at each of the time periods. The result of this type of experiment is what we now refer to as a one-step growth curve, which in principle, can be performed with any virus that can be propagated in cell culture (Fig. 2.4). The remarkable finding of this type of study was that infectious virus “disappeared” from the infected cultures for a variable period of time, depending on the virushost-cell system. This is referred to as the eclipse period, and represents the period of time that begins with cell entry/uncoating and ends with the appearance of newly formed infectious virus particles. Following the end of the eclipse period there is an essentially exponential increase in production of infectious virus particles until the host cell is unable to maintain its metabolic integrity.
Depending on the type of virus, there may be sudden release of virus particles following lysis of the host cell, as exemplified by T-even bacteriophages, or a prolonged release of virus particles via sustained budding of virus particles at a cell membrane site, such as with influenza A virus. The one-step growth curve can be used to divide the virus replication cycle into its component parts, which include attachment, the eclipse period (entry, uncoating, replication of component parts, virion assembly), and release of virus particles. Although the replication cycles of all conventional viruses follow these same general steps, the details of each step can vary widely depending on the specific virus. Therefore, the kinetics of the onestep growth curve differs with the unique properties of the specific virushost-cell system used. To ensure that all steps of the virus replication cycle are temporally synchronized, it is important that the infection be initiated with enough virus particles to simultaneously infect all cells in the culture. This is achieved by using a high multiplicity of infection [typically 10 plaque forming units (pfu) of virus/cell]. A discussion focusing on the individual steps of the general virus replication cycle now follows. This discussion includes expanded descriptions and details of complete replication cycles for model viruses representing four major groups [positive strand RNA viruses (picornavirus), negative strand RNA viruses (rhabdovirus), retroviruses, and DNA viruses (adenovirus)]. More comprehensive discussions covering the specific details of individual virus families are found in Part II of this book.
Attachment
The critical first step in the virus replication cycle is the attachment of the virus particle to a host cell. Attachment requires specific interactions between components of the virus particle (eg, capsid proteins or envelope glycoproteins) and components of the host cell (eg, a glycoprotein or carbohydrate moiety). This process can be conceptually simple whereby attachment can involve interactions between a single component of the virus with a single component of the cell. For example, binding of influenza A virus to a host cell requires only an interaction between the viral hemagglutinin (HA) glycoprotein and a sialic acid residue on the cell surface.
Alternatively, attachment-related interactions can be complex and involve sequential interactions between multiple components of both the virus and the cell. Examples of this type of cell binding are described below in the expanded discussions of the adenovirus and retrovirus replication cycles. Many host proteins are not widely expressed but instead are expressed in a cell- or tissue-specific manner. Therefore, receptor usage plays an important role in defining the tissue/organ specificity (tropism) of a virus. In turn, the tissue and organ specificity of a virus largely defines its pathogenic potential and the nature of the disease it causes. Similarly, cellular components (eg, proteins, carbohydrate structures, etc.) can differ markedly between organisms, thus, receptor usage also influences the types of organisms (host species) that a virus can infect (host range).
Virus particles interact with cell-surface molecules which are referred to as receptors, coreceptors, attachment factors, or entry factors depending on the role(s) that they play in the attachment and entry processes. Frequently, the term “viral receptor” is used to describe these cellsurface molecules, which is something of a misnomer, as cells certainly do not maintain receptors for the purpose of binding viruses. Rather, viruses have evolved to use host cell molecules that perform functions related to normal cellular processes. Initial contact of a virus particle with the cell surface often involves short-distance electrostatic interactions with charged molecules such as heparan sulfate proteoglycans. This initial contact may simply help to concentrate virus on the surface of the cell, which facilitates the establishment of more specific interactions with other receptor-like molecules. The affinity of binding between an individual virus component and its cellular ligand may be low; however, the virus surface possesses many receptor binding sites, thus the affinity of binding between the virus and the host cell is enhanced by the establishment of multiple virus/receptor interactions. Although viruses require at least one receptor to be expressed on the surface of the host cell, some viruses must also engage an intracellular receptor(s) in order to initiate a productive infection. These intracellular interactions do not play a role in attachment to the cell but instead are required for the final stages of the entry/ uncoating process; and therefore, will be discussed in more detail below. The identification of host cell factor(s) that serve as receptors for virus attachment is important for understanding the molecular details of specific virus replication cycles, and also has practical implications as this knowledge can inform the design of antiviral drugs. In recent years, numerous host cell components capable of functioning as receptors/entry factors for viruses have been identified. These include ligand-binding receptors (eg, chemokine receptors, transferrin receptor 1), signaling molecules (eg, CD4), cell adhesion/signaling receptors (eg, intercellular adhesion molecule-1, ICAM-1), enzymes, integrins, and glycoconjugates with various carbohydrate linkages, sialic acid being a common terminal residue (Table 2.1). As shown in Table 2.1, different viruses may use the same receptor/entry factor (eg, Coxsackievirus and some adenoviruses), which results in these viruses having a shared or overlapping cell/tissue tropism. The number and identity of host cell molecules that play a part in the initial interactions of virus with host cells will certainly increase as new viruses are identified and as existing viruses are better characterized. The process of identifying receptors/entry factors is more complicated than initially imagined, as viruses within a given family may use different receptors. Furthermore, different strains of the same virus can utilize different receptors and adaptation of a virus to growth in cell culture can change receptor usage of the virus. For example, wild-type strains of foot and mouth disease virus bind to integrins in vivo, but cell culture-passaged strains of the virus can use heparan sulfate. This change in receptor specificity alters the pathogenicity of the virus, clearly indicating that receptor usage influences the disease process. Some viruses with a broad host range, such as arthropod-borne viruses and some of the alphaherpesviruses, are thought to use several different host-specific receptors, which accounts for their ability to grow in cells from many hosts. Alternatively, a virus can use a common receptor that is expressed in multiple host species. For example, Sindbis virus was recently shown to utilize a protein called natural resistance-associated macrophage protein (NRAMP) as a receptor in insect cells, and to use the mammalian homolog (NRAMP2) for binding to cultured mammalian cells and in the tissues of mice. Two additional issues related to the virus/cell attachment process are notable. A model was recently proposed in which cell receptors that normally function in the recognition and clearance of apoptotic cells are used for cell attachment/entry by dengue virus and perhaps by related flaviviruses. Flaviviruses bud through the membrane of the endoplasmic reticulum, and consequently, are thought to incorporate phosphatidylserine (PtdSer) into the outer leaflet of the viral envelope. PtdSer is also enriched on the outer leaflet of the plasma membrane of cells undergoing apoptosis due to lipid reshuffling, and is bound directly or indirectly by members of the TIM and TAM families of transmembrane receptor proteins, respectively. TIM and TAM proteins are expressed by a number of cell types including macrophages and dendritic cells, which are normal targets of dengue virus infection. Under normal circumstances the binding between the TIM/TAM proteins on myeloid cells and the PtdSer on apoptotic cells leads to the uptake and clearance of the apoptotic cell. By incorporating PtdSer into the viral envelop, dengue virus is thought to mimic an apoptotic cell, enabling the virus to be bound and internalized by cells expressing the TIM/TAM proteins. This mechanism does not appear to be unique to flaviviruses as a similar model has been proposed for vaccinia virus (family Poxviridae). A second somewhat indirect mechanism of cell binding/entry is also best exemplified by dengue virus. This mechanism is referred to as antibody-dependent enhancement of infection, which occurs when the virus particle is bound by nonneutralizing IgG antibodies that in turn are bound by activating Fcγ receptors expressed on the surface of mononuclear phagocytic cells (eg, macrophages). This interaction leads to internalization of the antibody/virus complex and eventual release of infectious virus into the cytoplasm of the phagocytic cell. Foot and mouth disease virus and feline coronavirus can also infect cells through this antibody-mediated enhancement mechanism in vitro, but its importance in the natural infection process is conjectural.