MECHANISMS OF VIRAL INJURY AND DISEASE
The most common adaptation of a virus to a host involves infection, spread, and shedding with minimal if any adverse effects on the host. Medically relevant virus infections are distinct in that infection causes tissue injury and thus disease (Fig. 3.8).
FIGURE 3.8 Potential mechanisms by which steps in the virus replication cycle may cause injury to cells
Tissue injury may facilitate virus propagation within or transmission between hosts, and at minimum should not interfere with these processes if the virus is to be maintained within a specific population of animals.
Virus-induced cytopathic effects may induce inflammatory and physiological responses such as coughing and sneezing that facilitate shedding and transmission. Induction of diarrhea is another means of facilitating transmission by enhancing environmental contamination with progeny virus. Virus-induced immune suppression may confound host attempts at clearance and thus benefit viral spread, while also predisposing the infected host to secondary microbial infections. Tissue injury may reflect host defense mechanisms that include apoptosis or immune responses that target virus-infected cells.
In other instances, damage to the host may be a consequence of virus replication in which there is no known advantage to either the virus or host, or reflects a byproduct of infection with no significant impact on transmission. The latter includes many instances where viruses infect the CNS, resulting in congenital malformations in fetuses or neonates, or clinically significant inflammatory disease in older animals. Host species is a significant variable when considering the potential of a virus to cause disease, where a given virus may cause clinically inapparent infection in a reservoir species and clinical disease in a species to which the virus is less adapted. Mechanisms of virus-induced tissue injury may be considered “direct” when they are a direct consequence of virus replication within a cell or tissue, and “indirect” when the injury is mediated by a host immune or inflammatory response.
Types of Virus Cell Interactions
Virus-induced tissue injury reflects viral cell and tissue tropism, and the mode of replication within the infected cells. As described in the preceding section, cellular tropism of viruses is determined by the presence of appropriate cellular receptors and an environment that is conducive to virus gene expression and replication. The latter may include the expression of cell-type-specific proteases, transcription factors, and other factors required for viral replication. Cells are said to be permissive to infection if they provide such an environment. Viruses typically encode genes that modulate host-cell functions for their own benefit and, of course, the host has elaborate innate defenses to restrict viral functions (see Chapter 4: Antiviral Immunity and Virus Vaccines).
Permissiveness may thus also reflect the ability of a virus to inhibit innate antiviral defense mechanisms. Viral and cellular factors that influence the outcome of infection are often in delicate balance and easily shifted one way or the other. The dynamic nature of the virus cell relationship is defined in terms that describe the degree of damage to the infected cell and the production of viral progeny.
Cytopathic infections are characterized by loss of cell functions that are essential to survival. Cell degeneration and necrosis or virus-induced apoptosis are final outcomes of cytopathic infections. These infections are alternatively described as cytocidal (meaning “cell death”) or cytolytic (meaning “cell lysis” or “rupture”).
Cell lysis is required for release of nonenveloped viral progeny, whereas progeny of enveloped viruses can be released by budding from viable cells.
Cell maintenance functions are preserved in noncytopathic infections. Noncytopathic infections can be clinically significant when they disrupt cell specialized functions. For example, noncytopathic infections of neurons may cause loss of impulse conduction, and noncytopathic infection of oligodendrocytes may result in loss of myelin formation, both of which contribute to clinical neurological disease despite survival of the infected cells. A noncytopathic viruscell relationship may give rise to a persistent infection due to survival of the cell, the inability of immune mechanisms to eliminate the virus, and a low level of virus replication that assures persistence of the virus’ genetic information. Persistence may be associated with production of viral progeny (productive infections) or the absence of viral progeny (nonproductive infections), whereas cytopathic infections are generally productive. A persistent productive infection may result in viral carriers capable of lifelong shedding, and may continually seed infections within the host and stimulate immune and inflammatory responses that contribute to chronic disease.
Latent infection may be viewed as a type of persistent infection in which the viral genome is not transcribed and so there is no production of viral proteins or progeny. The viral genome is maintained indefinitely in the cell, either by the integration of the viral nucleic acid into the hostcell DNA or by carriage of the viral nucleic acid in the form of an episome, and the infected cell survives and may divide repeatedly. As such, latent infections are restricted to infection by DNA viruses or RNA viruses capable of generating DNA copies of their genome. Clinical significance of these infections is that virus gene expression can be periodically reactivated, giving rise to the production of viral protein and infectious viral progeny. This is the case of neurons latently infected with herpesviruses, where reactivation results in progeny production that in turn is amplified by productive cytopathic infections of other tissues. Persistent or latent infections with oncogenic viruses may also lead to cell transformation, as described later in this chapter. The various types of interaction that can occur between virus and cell are summarized in Table 3.2 and in Fig. 3.8.
Cytopathic Changes in Virus-Infected Cells
Cytopathic viral infections ultimately kill the cells in which they replicate, by preventing synthesis of host macromolecules (as described below), by producing degradative enzymes or toxic products, or by inducing apoptosis. In a productive infection of tissue culture cells, the first round of virus replication yields progeny virions that spread through the medium to infect both adjacent and distant cells; all cells in the culture may eventually become infected. Cells exhibit biochemical and structural changes that are collectively referred to as a cytopathic effect. Some cytopathic effects have a light microscopic appearance that is characteristic of the particular virus involved, and is therefore an important preliminary clue in the identification of clinical isolates in the diagnostic laboratory (see Chapters 2 and 5: Virus Replication and Laboratory Diagnosis of Viral Infections) (Fig. 3.9).
FIGURE 3.9 Cytopathic effects produced by viruses. Inclusions may reflect viral replication complexes in the nucleus or cytoplasm. Cell rounding may follow cytoskeletal disruption. Syncytia formation may be seen following infection with enveloped viruses. Apoptosis is a programmed cell death resulting in morphologic changes that are distinct from necrosis or lysis (ie, forms of nonprogrammed cell death), and is a form of host defense. A single virus may cause combinations of these cytopathic effects
Other changes reflect disruption of cellular processes that are less specific to the infecting virus. Apoptotic cells have a characteristic light microscopic appearance, although this host defense mechanism can be elicited by members of numerous virus families. Similarly, virusinduced metabolic and toxic insults to the cell may result in morphological changes indicative of cell degeneration and necrosis, the cumulative effect of numerous insults that may be triggered by a number of different viruses. Cytopathic effects should be viewed in context of their relationship to viral replication; to what degree is the change unique to a particular group of viruses, and does the change influence the virus’ ability to produce progeny?
Inclusion bodies are sites of viral transcription and genome replication in the cell that are readily apparent in cells by light microscopy. DNA viruses that replicate in the nucleus utilize cell machinery to varying degrees in support of transcription and genome replication. Host cell DNA may be displaced from the nuclear matrix by the viral genome, resulting in chromatin margination along the nuclear membrane as aggregates of viral nucleic acid and protein accumulate. The result is an intranuclear inclusion—an aggregate of uniform staining that is distinct from nuclear structures observed in uninfected cells. Stains used routinely in diagnostic settings yield red signal for protein, and blue signal for nucleic acid. Intranuclear inclusions typically stain red, indicative of the high viral protein content, whereas the marginalized chromatin is blue. Intranuclear inclusions are characteristic of cells infected with herpesviruses and adenoviruses.
Occasionally an RNA virus will induce structures known as nuclear bodies, a type of intranuclear inclusion that is host in origin but rich in viral protein. These structures are thought to regulate RNA processing within the cell, and are the basis for the intranuclear inclusions of canine distemper virus infection. Cytoplasmic inclusions are typical of viruses replicating to high levels in the cytoplasm, again reflecting aggregates of viral genomes engaged in transcription and replication. Cytoplasmic inclusions are typical of infections caused by poxviruses, paramyxoviruses, rhabdoviruses, and reoviruses (Fig. 2.2). The diagnostic importance of these structures is illustrated by the fact that some of these inclusions are known by specific names, such as the “Negri bodies” of rabies virus infected neurons.
Inhibition of Host Cell Protein Production while viral protein synthesis continues is a characteristic of many viral infections. This shutdown is particularly rapid and profound in picornavirus infections, but it is also pronounced in togavirus, influenza virus, rhabdovirus, poxvirus, and herpesvirus infections. With some other viruses, the shutdown occurs late in the course of infection and is more gradual, whereas with noncytocidal viruses, such as pestiviruses, arenaviruses, and retroviruses, there is no dramatic inhibition of host-cell protein synthesis, and no cell death. Viruses have evolved numerous mechanisms to interfere with host-cell mRNA transcription, processing, and translation. Inhibition of both host-cell DNA replication and mRNA transcription is a consequence of DNA virus infection when cellular machinery is redirected to viral templates. Inhibition may reflect a broader strategy by the virus to preserve nucleotide pools in support of virus replication, and to diminish cellular mRNA levels that would otherwise compete with viral mRNA for translational machinery. This phenomenon is observed during replication of viruses in several different families, including poxviruses, rhabdoviruses, reoviruses, paramyxoviruses, and picornaviruses. In some instances, this inhibition may be the indirect consequence of viral effects on host-cell protein synthesis that decrease the availability of transcription factors required for DNA-dependent RNA polymerase activity. Inhibition of processing and translation of host-cell messenger RNA occurs during replication of vesicular stomatitis viruses, influenza viruses, and herpesviruses, through interference with the splicing of cellular primary mRNA transcripts that are needed to form mature mRNAs. In some instances, spliceosomes are formed, but subsequent catalytic steps are inhibited. For example, a protein synthesized in herpesvirus-infected cells suppresses RNA splicing and leads to reduced amounts of cellular mRNAs and the accumulation of primary mRNA transcripts. In addition to interference with host-cell mRNA transcription and processing, viruses may produce factors that bind to ribosomes and inhibit cellular mRNA translation. Viral proteins may inhibit the processing and transport of cellular proteins from the endoplasmic reticulum, and this inhibition may lead to their degradation. This effect is seen in lentivirus and adenovirus infections. Influenza viruses remove the 5’ cap structure of cellular mRNAs to initiate synthesis of viral mRNAs, the cap being required for translation. Other viruses simply produce viral mRNAs in large quantities in order to assure translation, outcompeting cellular mRNAs for cellular translation machinery by mass action. The cumulative effect of inhibition of host-cell protein synthesis and depletion of nucleotide pools can be the loss of cellular homeostasis, resulting in a sequence of degeneration and necrosis. This progression is relatively nonspecific as to cause, with similar changes being induced by physical or chemical insults. The most common early and potentially reversible change is cloudy swelling, a change associated with increasing permeability of the cellular membranes leading to swelling of the nucleus, distention of the endoplasmic reticulum and mitochondria, and rarefaction of the cytoplasm. Later in the course of many viral infections the nucleus becomes condensed and shrunken, and cytoplasmic density increases. Cell destruction can be the consequence of further loss of osmotic integrity and leakage of lysosomal enzymes into the cytoplasm. This progression is consistent with the so-called common terminal pathway to cell death. In contrast to these nonspecific changes are toxicities induced by viral proteins that interfere with cellular membrane or cytoskeletal structure and function.
Interference with Cellular Membrane Function can affect the participation of cellular membranes in many phases of virus replication, from virus attachment and entry, to the formation of replication complexes, to virion assembly. Viruses may alter plasma membrane permeability, affect ion exchange and membrane potential, or induce the synthesis of new intracellular membranes or the rearrangement of previously existing ones.
For example, a generalized increase in membrane permeability occurs early during picornavirus, alphavirus, reovirus, rhabdovirus, and adenovirus infections. Early changes in cell structure often are dominated by proliferation of various cell membranes: for example, herpesviruses cause increased synthesis, even reduplication, of nuclear membranes; flaviviruses cause proliferation of the endoplasmic reticulum; picornaviruses and caliciviruses cause a distinctive proliferation of vesicles in the cytoplasm; and many retroviruses cause peculiar fusions of cytoplasmic membranes.
Enveloped viruses specifically direct the insertion of their surface glycoproteins, including fusion proteins, into host-cell membranes as part of their budding process, and this may lead to membrane fusion between infected and uninfected cells, resulting in the formation of a multinucleated syncytium. This activity is restricted to enveloped viruses whose fusion proteins are activated when viral membrane glycoproteins come in contact with a cellular receptor. Normally this process allows fusion of the virion envelope with the cytoplasmic membrane of a target cell during the initiation of an infection, allowing entry the viral genome into the cytoplasm. In the course of virus replication however, these same fusion proteins are inserted into the cytoplasmic membrane of the infected cell in preparation for viral budding (Fig. 3.10).
FIGURE 3.10 Formation of syncytia by enveloped viruses. Following enveloped virus attachment and penetration, the genome is transcribed to produce the envelope proteins that are essential to these processes: attachment to and fusion between the viral envelope and the cell membrane. These viral proteins are inserted into the cell membrane in preparation for viral assembly (budding), but if they contact a neighboring uninfected cell, they can mediate attachment to and fusion with the membranes of that neighboring cell. The result is a multinucleated syncytium.
If viral membrane glycoproteins engage receptors on neighboring cells, the fusion proteins may be activated to cause fusion of one cell membrane with another, giving rise to the syncytial cell. Syncytia are a conspicuous feature of infection of cell monolayers in culture by lentiviruses, coronaviruses, paramyxoviruses, and some herpesviruses. Syncytia may also be observed in tissue of infected animals, particularly for paramyxoviruses; for example, in horses infected with Hendra virus and cattle infected with respiratory syncytial virus. Syncytium formation has been suggested as a means by which viruses spread in tissues: fusion bridges may allow subviral entities, such as viral nucleocapsids and nucleic acids, to spread while avoiding host defenses. Relevance of this mechanism is likely limited to specific cell types, being implicated as a means for neuron-to-neuron spread of rhabdoviruses and paramyxoviruses where fusion events are likely restricted to very small cell contact points within the synapse. Viral proteins (antigens) inserted into the host-cell plasma membrane may constitute targets for specific humoral and cellular immune responses that cause the lysis of the infected cell. This may happen before significant progeny virus is produced, thus slowing or arresting the progress of infection and hastening recovery (see Chapter 4: Antiviral Immunity and Virus Vaccines). It is for this reason that accumulation of viral membrane glycoproteins occurs late in the infection cycle, in preparation for viral budding. Although these viral proteins are attractive targets for immune clearance, the host response may also contribute to immune-mediated tissue injury and disease. Viral antigens may also be incorporated in the membrane of cells transformed by viruses, and play an important role in immune-mediated resolution or regression of viral papillomas, for example. It should be noted that cell surface expression of membrane glycoproteins was exploited early in the development of diagnostic tests (see Chapter 5: Laboratory Diagnosis of Viral Infections). Many of these viral proteins have the potential to bind glycoproteins expressed on the surface of red blood cells in a species specific manner. Cells in monolayer cultures infected with influenza viruses, paramyxoviruses, and togaviruses, all of which bud from the plasma membrane, acquire the ability to adsorb erythrocytes in a phenomenon termed hemadsorption (Fig. 2.1D; Fig. 3.11). The viral membrane glycoprotein serves as a receptor for ligands on the surface of erythrocytes. The same glycoprotein spikes are responsible for hemagglutination in vitro—that is, the agglutination of erythrocytes by free viral particles. Hemadsorption and hemagglutination are not known to play a part in the pathogenesis of viral diseases.
Disruption of the Cell Cytoskeleton causes changes in cell shape (eg, rounding) that are characteristic of many viral infections. The cytoskeleton is made up of several filament systems, including microfilaments (eg, actin), intermediate filaments (eg, vimentin), and microtubules (eg, tubulin). The cytoskeleton is responsible for the structural integrity of the cell, for the transport of organelles through the cell, and for certain cell motility activities. Particular viruses may damage specific filament systems: for example, canine distemper virus, vesicular stomatitis viruses, vaccinia virus, and herpesviruses cause a depolymerization of actin-containing microfilaments, and enteroviruses induce extensive damage to microtubules. Such damage contributes to the drastic cytopathic changes that precede cell lysis in many infections. The elements of the cytoskeleton are also employed by many viruses in the course of their replication: in virus entry, in the formation of replication complexes and assembly sites, and in virion release.
Apoptosis is the process of programmed cell death, which is essentially a mechanism of cell suicide that the host activates as a last resort to eliminate viral factories before progeny virus production is complete. It was long thought that viruses killed cells exclusively by direct means such as usurping their cellular machinery or disrupting membrane integrity, ultimately leading to necrosis of the virus-infected cell. However, it is now clear that apoptosis is an important and common event during many viral infections. There are two distinct cellular pathways that trigger apoptosis, both of which culminate in the activation of host-cell caspase enzymes that mediate death of the cell (the so-called executioner phase). Once activated, caspases are responsible for degradation of the cell’s own DNA and proteins. Cell membrane alterations in the doomed cell promote its recognition and removal by phagocytic cells. The two initiation pathways are:
1. The Intrinsic (Mitochondrial) Pathway. The mitochondrial pathway is activated as a result of increased permeability of mitochondrial membranes subsequent to cell injury, such as that associated with a viral infection. Severe injury alters the delicate balance between antiapoptotic (eg, Bcl-2) and proapoptotic (eg, Bax) molecules in mitochondrial membranes and the cytosol, resulting in progressive leakage of mitochondrial proteins (such as cytochrome c) into the cytosol where these proteins activate cellular caspases.
2. The Extrinsic (Death Receptor) Pathway. The extrinsic pathway is activated by engagement of specific cell-membrane receptors, which are members of the tissue necrosis factor (TNF) receptor family (TNF, Fas, and others). Thus binding of tissue necrosis factor to its cellular receptor can trigger apoptosis. Similarly, cytotoxic T lymphocytes that recognize virus-infected cells in an antigen-specific manner can bind the Fas receptor, activate the death domain, and trigger the executioner caspase pathway that then eliminates the cell before it becomes a functional virus factory.
Noncytopathic Changes in Virus-Infected Cells
Noncytopathic viral infections usually do not kill the cells in which replication occurs. On the contrary, they often cause persistent infection during which infected cells produce and release virions but cellular metabolism that is essential to maintaining homeostasis is either not affected or is minimally affected. In many instances, infected cells even continue to grow and divide. This type of interaction can occur in cells infected with several kinds of RNA viruses, notably pestiviruses, arenaviruses, retroviruses, and some paramyxoviruses. Nevertheless, with few exceptions (eg, some retroviruses), there are slowly progressive changes that ultimately lead to cell death. In the host animal, cell replacement occurs so rapidly in most organs and tissues that the slow fallout of cells as a result of persistent infection may have no effect on overall function, whereas terminally differentiated cells such as neurons, once destroyed, are not replaced, and persistently infected differentiated cells may lose their capacity to carry out specialized functions. Viruses such as the pestiviruses, arenaviruses, Bornavirus, and retroviruses that do not shut down hostcell protein, RNA, or DNA synthesis and that do not rapidly kill their host cells, can produce important pathophysiologic changes in their hosts by affecting crucial functions that are associated neither with the integrity of cells nor their basic housekeeping functions. Damage to the specialized functions of differentiated cells may still affect complex regulatory, homeostatic, and metabolic functions, including those of the central nervous system, endocrine glands, and immune system.
Virus-Mediated Tissue and Organ Injury
The severity of a viral disease is not necessarily correlated with the degree of cytopathology produced by the causative virus in cells in culture. Many viruses that are cytocidal in cultured cells do not produce clinical signs in vivo (eg, many enteroviruses), whereas some that are noncytocidal in vitro cause lethal disease in animals (eg, rabies virus). Further, depending on the organ affected, cell and tissue damage can occur without producing clinical signs of disease—for example, a large number of hepatocytes (liver cells) may be destroyed in Rift Valley fever in sheep without obvious clinical signs. When damage to cells does impair the function of an organ or tissue, this may be relatively insignificant in a tissue such as skeletal muscle, but potentially devastating in organs such as the heart or the brain. Likewise, virus-induced inflammation and edema are especially serious consequences in organs such as the lungs and CNS
Mechanisms of Viral Infection and Injury of Target Tissues and Organs
The mechanisms by which individual viruses cause injury to their specific target organs are described in detail under individual virus families in Part II of this book, thus the objective of this section is to provide a brief overview of potential pathogenic mechanisms that viruses can use to cause injury in their target tissues.
Viral Infection of the Respiratory Tract
Viral infections of the respiratory tract are extremely common, especially in animals housed in crowded settings. Individual viruses exhibit tropism for different levels of the respiratory tract, from the nasal passages to the pulmonary airspaces (terminal airways and alveoli), but there is considerable overlap. Tropism of respiratory viruses is probably a reflection of the distribution of appropriate receptors and intracellular transcriptional enhancers, as well as physical barriers, physiological factors, and immune parameters. For example, bovine rhinitis viruses (Family Picornaviridae) replicate in the nasal passages because their replication is optimized at lower temperatures, whereas bovine respiratory syncytial virus (Family Paramyxoviridae) preferentially infects epithelial cells lining the terminal airways; thus rhinitis viruses may cause mild rhinitis, whereas respiratory syncytial virus is the cause of bronchiolitis and bronchointerstitial pneumonia. Some viruses cause injury to the type I or type II pneumocytes lining the alveoli, either directly or indirectly; if extensive, injury to type I pneumocytes leads to acute respiratory distress syndrome, whereas injury to type II pneumocytes delays repair and healing in the affected lung. Influenza viruses replicate in both the nasal passages and airways of infected mammals, but influenza virus infection is typically confined to the lung because of the requirement for hemagglutinin cleavage by tissue-specific proteases. However, highly virulent influenza viruses such as the EurasianAfrican H5N1 virus can spread beyond the lungs to cause severe generalized (systemic) infection and disease. The ability of this virus to escape the lung may be related to its tropism for type I pneumocytes that line alveoli, and its ability to cause systemic disease may reflect the fact that its hemagglutinin can be cleaved by proteases that are present in many tissues. Similarly in birds, high-pathogenicity avian influenza viruses have several basic amino acids at the hemagglutinin cleavage site, which can be cleaved intracellularly by ubiquitous endopeptidase furins located in the trans-Golgi network in a wide variety of cell types in various tissues. In contrast, the hemagglutinin protein of low pathogenicity avian influenza viruses is cleaved extracellularly by tissue-restricted proteases that are confined to the respiratory and gastrointestinal tracts (see Chapter 21: Orthomyxoviridae). Regardless of the level of the respiratory tree that is initially infected, viral infection typically leads to local cessation of cilial activity, focal loss of integrity of the lining mucus layer, and multifocal destruction of small numbers of epithelial cells (Fig. 3.12). Initial injury is followed by progressive infection of epithelial cells within the mucosa, and inflammation of increasing severity, with exudation of fluid and influx of inflammatory cells. Fibrin-rich inflammatory exudate and necrotic cellular debris (degenerate neutrophils and sloughed epithelium) then accumulate in the lumen of the affected airways or passages, with subsequent obstruction and, in severe cases, increasing hypoxia and respiratory distress. The mucosa is quickly regenerated in animals that survive, and adaptive immune responses clear the infecting virus and prevent reinfection for variable periods of time (depending on the particular virus). In addition to their direct adverse consequences, viral infections of the respiratory tract often predispose animals to secondary infections with bacteria, even those bacteria that constitute the normal flora in the nose and throat. This predisposition can result from interference with normal mucociliary clearance as a consequence of viral injury to the mucosa, or suppression of innate immune responses. For example, cellular expression of Toll-like receptors is depressed in the lung after influenza virus infection, and thus convalescent animals may be less able to quickly recognize and neutralize invading bacteria. This potential synergy between respiratory viruses and bacteria is compounded by overcrowding of animals as occurs during shipping and in feedlots and shelters. Environmental factors may combine to facilitate concurrent airway infection by multiple viruses and bacteria. These polymicrobial infections are facilitated by the immunosuppressive effects of stress, the induction of ciliary stasis by exposure to ammonia vapor from animal waste, and crowded humid environments that facilitate aerosol transmission of enveloped viruses.
Viral Infection of the Gastrointestinal Tract
Infection of the gastrointestinal tract can be acquired either by ingestion of an enteric virus (eg, rotaviruses, coronaviruses, astroviruses, etc.) where infection is confined to the gastrointestinal tract or as a consequence of generalized hematogenous spread during a systemic viral infection such as with certain parvoviruses (eg, feline panleukopenia, canine parvovirus), pestiviruses (eg, bovine viral diarrhea virus), and morbilliviruses (eg, canine distemper and rinderpest viruses). Enteric viral infections usually result in rapid onset of gastrointestinal disease after a short incubation period, whereas systemic infections have a longer incubation period and are typically accompanied by clinical signs that are not confined to dysfunction of the gastrointestinal tract. Virus-induced diarrhea is a result of infection of the epithelial cells (enterocytes) lining the gastrointestinal mucosa. Rotaviruses, astroviruses, and enteric coronaviruses characteristically infect the more mature enterocytes that line the intestinal villi, whereas parvoviruses and pestiviruses infect and destroy the immature and dividing enterocytes present in the intestinal crypts. Regardless of their site of predilection, these infections all destroy enterocytes in the gastrointestinal mucosa and so reduce its absorptive surface, leading to malabsorption diarrhea with attendant loss of both fluid and electrolytes. The pathogenesis of enteric virus infections can be even more complex than simple virus-mediated destruction of enterocytes; for example, rotaviruses produce a protein (nsp4) that itself causes secretion of fluid into the bowel (intestinal hypersecretion), even in the absence of substantial virus-mediated damage. In suckling neonates, undigested lactose from ingested milk passes through the small bowel to the large bowel, where it exerts an osmotic effect that further exacerbates fluid loss. Neonates are also disadvantaged by the fact that the replacement rate of enterocytes is not as high as in older animals. Animals with severe diarrhea can rapidly develop pronounced dehydration, hemoconcentration, acidosis that inhibits critical enzymes and metabolic pathways, hypoglycemia, and systemic electrolyte disturbances (typically, decreased sodium and increased potassium), and diarrhea can be quickly fatal in very young or otherwise compromised animals. Enteric virus infections that occur via the oral route generally begin in the stomach or proximal small intestine, and they then spread caudally as a “wave” that sequentially affects the jejunum, ileum, and large bowel. As the infection progresses through the bowel, absorptive cells destroyed by the infecting virus are quickly replaced by immature enterocytes from the intestinal crypts. The presence of increased numbers of these immature enterocytes contributes to malabsorption and intestinal hypersecretion (fluid and electrolyte loss). Adaptive immune responses lead to mucosal IgA and systemic IgG production in animals that survive, conferring resistance to reinfection. Enteric virus infections in neonates are frequently associated with infections by other enteric pathogens, including bacteria (eg, enterotoxigenic or enteropathogenic Escherichia coli) and protozoa such as Cryptosporidium spp., probably because of the common factors (crowding, poor sanitation) that predispose to these infections.
Viral Infection of the Skin
In addition to being a site of initial infection, the skin may be invaded secondarily via the bloodstream. Thus skin lesions that accompany viral infections can be either localized, such as papillomas, or disseminated. In animals, erythema (reddening) of the skin as a consequence of systemic viral infections is most obvious on exposed, hairless, nonpigmented areas such as the snout, ears, paws, scrotum, and udder. In addition to papillomas (warts, see Chapter 11: Papillomaviridae and Polyomaviridae), virus-induced lesions that commonly affect the skin of virus-infected animals include macules (flat discolored areas of skin), papules (raised areas of skin), vesicles (fluid-filled raised areas of skin), and pustules (raised areas of skin containing leukocytes). Viruses of particular families tend to produce characteristic cutaneous lesions, frequently in association with similar lesions in the oral and nasal mucosa, the teats and genitalia, and at the junction of the hooves and skin of ungulates. Vesicles are especially important cutaneous lesions that are characteristic of important, potentially reportable diseases of livestock; in particular, vesicle formation is characteristic of foot-and-mouth disease and other viral diseases that can mimic it, although vesicles clearly can occur in other diseases not caused by viruses. Vesicles are essentially discrete “blisters” that result from accumulation of edema fluid within the affected epidermis, or separation of the epidermis from the underlying dermis (or mucosal epithelium from the submucosa). Vesicles rupture quickly to leave focal ulcers. Papules are either localized (eg, orf) or disseminated (eg, lumpy skin disease) epithelial proliferations that are characteristic of poxvirus infections. These proliferative and raised lesions frequently become extensively encrusted with inflammatory exudate. Virus infections that result in widespread endothelial injury in blood vessels throughout the body, including those of the subcutaneous tissues, can produce subcutaneous edema and erythema or hemorrhages in the skin and elsewhere (including the oral cavity and internal organs).
Viral Infection of the Central Nervous System
The CNS (brain and spinal cord) is exquisitely susceptible to serious, often fatal injury by certain viral infections provided the virus can gain access to these tissues. Viruses can spread from distal sites to the brain via nerves (Fig. 3.7), or via the blood (Fig. 3.13). Spread via nerves may involve peripheral nerve endings or infection of olfactory neurons in the nasal cavity with subsequent viral transport by axons of the olfactory nerve directly into the brain. To spread from the blood, viruses must cross either the bloodbrain or bloodcerebrospinal fluid barriers. The bloodbrain barrier consists of endothelial cells that are nonfenestrated and connected by tight junctions, which in turn are surrounded by a basement membrane and astrocytes. Viruses may cross this barrier by either direct infection of endothelial cells and spread of infection to the adjacent astrocytes, or the virus may be carried across the barrier by infected leukocytes that are engaged in immune surveillance of the CNS or inflammatory responses. The bloodcerebrospinal fluid barrier is formed by tight junctions between epithelial cells of the choroid plexus, which are highly vascular structures producing the cerebrospinal fluids that circulate within the ventricles of the brain, the central canal of the spinal cord, and the leptomeninges that cover the surface of both brain and spinal cord. There is no barrier between the bloodstream and the epithelial cells of the plexus, and if virus can infect the choroid plexus epithelial cells, it may be shed through the cerebrospinal fluid pathways to be widely disseminated in the CNS. Collectively, a virus’ ability to overcome these barriers and initiate CNS infection are known as neuroinvasiveness. Once present within the CNS, a number of viruses can quickly spread to cause progressive infection of neurons and/or glial cells (astrocytes, microglia, and oligodendrocytes). This capability is known as neurovirulence. A virus can be poorly neuroinvasive, but if infection is initiated, exhibit a high degree of neurovirulence. Cytopathic infections of neurons, whether caused by togaviruses, flaviviruses, herpesviruses, or other viruses, leads to encephalitis or encephalomyelitis characterized by neuronal necrosis, phagocytosis of neurons (neuronophagia), and perivascular infiltrations of inflammatory cells (perivascular cuffing). The small vessels of the meninges are frequently involved in virus-induced inflammation of the CNS, either alone (meningitis) or in combination with inflammation of the brain and spinal cord (meningoencephalitis and meningomyelitis). In contrast, virulent rabies virus infection of neurons is noncytocidal and evokes little inflammatory reaction, but it is uniformly lethal for most mammalian species. Other characteristic pathologic changes are produced by various viruses, and by prions that cause slowly progressive diseases of the CNS. In bovine spongiform encephalopathy in cattle and scrapie in sheep, for example, there is slowly progressive neuronal degeneration and vacuolization. In contrast, infection of glial cells in dogs with canine distemper leads to progressive demyelination. In most cases, infection of the CNS seems to be a dead end in the natural history of viruses—shedding and transmission does not occur, particularly when the infection is highly cytopathic. Viruses that successfully use neurons for transmission are in the minority and they typically exhibit noncytopathic or poorly cytopathic infections. Noncytopathic infection of neurons is needed for rabies virus to complete the cycle of infection within a host. Rabies is reliant upon axon transporters of viable cells to travel from the point of inoculation to the CNS, to spread within the CNS, and to spread from the CNS to peripheral organs such as salivary glands that amplify and shed progeny virus. Cell death and the attending inflammatory response can prevent the virus from completing this cycle in more highly cytopathic infections, where the virus is less adapted to its host. The alphaherpesviruses undergo latent infection of peripheral nerves, specifically the dorsal root ganglion neurons. When reactivated, the infection is productive yet noncytopathic for the neuron. Progeny infect epithelial cells of mucosal surfaces where the infection is both productive and cytopathic. All in all, it seems anomalous that neurotropism should be the outstanding characteristic of so many of the most notorious pathogens of animals and zoonotic pathogens of humans, and yet be the characteristic least related to virus propagation in nature.
Viral Infection of the Hemopoietic System
The hemopoietic system includes: (1) the myeloid tissues, specifically the bone marrow and cells derived from it—erythrocytes, platelets, monocytes, and granulocytes; and (2) the lymphoid tissues, which include the thymus, lymph nodes, spleen, mucosa-associated lymphoid tissues and, in birds, the cloacal bursa. Cells that populate the myeloid and lymphoid systems, including lymphocytes, dendritic cells, and cells of the mononuclear phagocytic system (monocytes and macrophages) are all derived from bone marrow (or equivalent hemopoietic tissue) precursors. It is therefore convenient to group these cells and tissues under the heading of the hemopoietic system and to dispense with historical terminology such as “lymphoreticular” or “reticuloendothelial” systems. Importantly, lymphocytes and mononuclear phagocytes (blood monocytes, tissue macrophages, dendritic cells) are responsible for adaptive immunity (see Chapter 4: Antiviral Immunity and Virus Vaccines), thus viral infections of these cells can have profound effects on immunity. Infection and damage to mononuclear phagocytes can inhibit both the innate and adaptive immune response to the virus, in addition to serving as a source of progeny virions. Some of the most destructive and lethal viruses known exhibit this tropism: filoviruses, arenaviruses, hantaviruses, orbiviruses such as African horse sickness and bluetongue viruses, certain bunyaviruses such as Rift Valley fever virus, alphaviruses such as Venezuelan equine encephalitis virus, and flaviviruses such as yellow fever virus. After initial invasion, infection with these viruses begins with their uptake by dendritic cells and/or macrophages in lymphoid tissues (lymph nodes, thymus, bone marrow, Peyer’s patches, and the white pulp of the spleen). Viral infection can then spread in these tissues, frequently leading to cytolysis of adjacent lymphocytes and immune dysfunction. Viral infections can result in either specific acquired immunodeficiency or generalized immunosuppression. A relevant example of this phenomenon is provided by infection of the cloacal bursa (bursa of Fabricius) in chickens (the site of B cell differentiation in birds) with infectious bursal disease virus, which leads to atrophy of the bursa and a severe deficiency of B lymphocytes, equivalent to bursectomy. The result is an inability of severely affected birds to develop antibody-mediated immune responses to other infectious agents, which in turn leads to an increase in susceptibility to bacterial infections such as those caused by Salmonella spp. and E. coli, and other viruses. Acquired immunodeficiency syndrome (AIDS) in humans is caused by the human immunodeficiency virus (HIV), and similar viruses infect monkeys (simian immunodeficiency viruses), cattle (bovine immunodeficiency virus), and cats (feline immunodeficiency virus). In susceptible animals, these viruses individually can infect and destroy specific but different cells of the immune system, thereby causing immunosuppression of different types and severity. Many other viruses (eg, classical swine fever virus, bovine viral diarrhea virus, canine distemper virus, feline and canine parvoviruses) that cause systemic infections, especially those that infect mononuclear phagocytes and/ or lymphocytes, may temporarily but globally suppress adaptive immune responses, both humoral and cellmediated. Affected animals are predisposed to diseases caused by other infectious agents during the period of virus-induced immunosuppression, a phenomenon that can also occur following vaccination with certain liveattenuated vaccines. The immune response to unrelated antigens may be reduced or abrogated in animals undergoing such infections. Virus-induced immunosuppression may in turn lead to enhanced virus replication, such as the reactivation of latent herpesvirus, adenovirus, or polyomavirus infections. Similarly, immunosuppression associated with administration of cytotoxic drugs or irradiation for chemotherapy or organ transplantation can predispose to recrudescence of herpesviruses and, potentially, others.
Viral Infection of the Fetus
Most viral infections of the dam do not lead to infection of the fetus due to barrier functions provided by the placenta, although severe infections of the dam can sometimes lead to fetal death and expulsion (abortion) in the absence of fetal infection. However, some viruses can cross the placenta to infect the fetus (Table 3.3). Such infections occur most commonly in young dams (such as first-calf heifers) that are exposed during pregnancy to pathogenic viruses to which they have no immunity, as a consequence of lack of either appropriate vaccination or previous natural infection. The outcome of fetal viral infection is dependent upon the properties (virulence and tropism) of the infecting virus, as well as the gestational age of the fetus at the time of infection. Severe cytolytic infections of the fetus, especially in early gestation, are likely to cause fetal death and resorption or abortion, which also is dependent on the species of animal affected—abortion is especially common in those species in which pregnancy is sustained by fetal production of progesterone (such as sheep), whereas pregnancy is less likely to be terminated prematurely in multiparous species in which pregnancy is maintained by maternally derived progesterone (such as swine). Teratogenic viruses are those that can cause developmental defects after in utero infection. The outcome of infections of pregnant animals with teratogenic viruses is influenced to a great extent by gestational age, which influences stages of organogenesis, degree to which biological barriers have formed in tissues such as the CNS, and degree of immune competence. Viral infections that occur during critical stages of organogenesis in the developing fetus can have devastating consequences from virus-mediated infection and destruction of progenitor cells before they can populate organs such as the brain. For example, Akabane, Cache Valley and Schmallenberg viruses, bovine viral diarrhea virus, and bluetongue virus can all cause teratogenic brain defects in congenitally infected ruminants, as can parvovirus infections in cats. Although immune competence generally is developed by mid-gestation, viral infections before this time can lead to a weak and ineffectual immune response that leads to persistent postnatal infection, such as persistent bovine viral diarrhea virus infection in cattle and congenital lymphocytic choriomeningitis virus infection in mice.
Viral Infection of Other Organs
Almost any organ may be infected with one or another kind of virus via the bloodstream, but most viruses have well-defined organ and tissue tropisms that reflect the factors described earlier (presence of receptors, intracellular and other physiological or physical determinants of infection). The clinical importance of infection of various organs and tissues depends, in part, on their role in the physiologic well-being of the animal. In addition to the organs and tissues already described (respiratory tract, gastrointestinal tract, skin, brain and spinal cord, hemopoietic tissues), viral infections of the heart and liver can also have especially devastating consequences. The liver is the target of relatively few viral infections of animals, in marked contrast to the numerous hepatitis viruses (hepatitis A, B, and C viruses in particular) and other viruses (eg, yellow fever virus) that are important causes of severe liver disease in humans. In animals, Rift Valley fever virus, mouse hepatitis virus, and infectious canine hepatitis virus characteristically affect the liver, as do several abortigenic herpesviruses after fetal infections (eg, infectious bovine rhinotracheitis virus, equine herpesvirus 1, pseudorabies virus). Virus-mediated cardiac injury is relatively uncommon in animals, but is characteristic of bluetongue and some other endotheliotrophic viral infections, and alphavirus infections of Atlantic salmon and rainbow trout. Viruses that cause widespread vascular injury can result in disseminated hemorrhages and/or edema as a result of increased vascular permeability. Vascular injury in these so-called hemorrhagic viral fevers can result either from viral infection of endothelial cells or the systemic release of vasoactive and inflammatory mediators such as tissue necrosis factor from other infected cells— particularly mononuclear phagocytes and dendritic cells. Viruses causing vascular injury include dengue virus, yellow fever virus, ebola virus, and different hantavirus infections in humans, and bluetongue and African horse sickness viruses in livestock. Widespread endothelial injury leads to thrombosis that may precipitate disseminated intravascular coagulation, which is the common pathway that leads to death of animals and humans infected with a variety of viruses that directly or indirectly cause vascular injury. Paradoxically, these infected individuals bleed profusely due to the consumption of clotting factors.
Nonspecific Pathophysiological Changes in Viral Diseases
Some of the adverse consequences of viral infections cannot be attributed to direct cell destruction by the virus, to immunopathology, or other physiological responses that may include release of endogenous adrenal glucocorticoids in response to the stress of the infection. Viral diseases are accompanied frequently by a number of vague general clinical signs, such as fever, malaise, anorexia, and lassitude. Cytokines (interleukin-1 in particular) produced in the course of innate immune responses to infection may be responsible for some of these signs, which collectively can significantly reduce the animal’s performance. Less characterized are the potential neuropsychiatric effects of persistent viral infection of particular neuronal tracts, such as that caused by Borna disease virus. Borna disease virus infection is not lytic in neurons, but induces bizarre changes in the behavior of rats, cats, and horses.
Virus-Induced Immunopathology
The adaptive immune response (eg, antibodies and cytotoxic T cells) to viruses could theoretically be harmful if the elimination of virusinfected cells leads to dangerous physiological consequences (eg, damage to liver or heart). The concept of virus-induced immunopathology is based on experimental results obtained in mouse models. Antibody-mediated immunopathology (also called type III hypersensitivity reactions) is caused by deposition of complexes of antigen and antibody (immune complexes) that initiate inflammation and tissue damage. Immune complexes circulate in blood in the course of most viral infections. The fate of the immune complexes depends on the ratio of antibody to antigen. The virus is typically cleared by tissue macrophages in infections where there is a large excess of antibody as compared with circulating virus, or even if there are equivalent amounts of antibody and virus. However, in some persistent infections, viral proteins (antigens) and/or virions are released continuously into the blood but the antibody response is weak and antibodies are of low avidity. In these instances, immune complexes are deposited in small blood vessels that function as filters, especially those of the renal glomeruli. Immune complexes continue to be deposited in glomeruli over periods of weeks, months, or even years, leading to their accumulation and subsequent immune-complex mediated glomerulonephritis. This phenomenon is observed in Aleutian mink disease (parvovirus infection), feline leukemia, and equine infectious anemia. A similar pathogenesis may underlie the progression of feline infectious peritonitis, a multisystemic disease associated with coronavirus infection in cats. T cell-mediated immunopathology (also called type IV reactions or delayed hypersensitivity reactions) has only been unequivocally demonstrated in mouse models of lymphocytic choriomeningitis virus infection.
Viruses and Autoimmune Disease
It has been proposed, with little definitive evidence, that viral infections may be responsible for autoimmune diseases in animals and humans. Proposed mechanisms for this largely hypothetical phenomenon focus on either unregulated or misdirected immune responses precipitated by a viral infection, or the presence of shared or equivalent antigens on infectious agents and host cells (molecular mimicry). Molecular mimicry clearly is responsible for immune-mediated diseases initiated by microbial infection, as classically illustrated by rheumatic heart disease in humans that is initiated by group A Streptococcus infection. In viruses, individual epitopes have been identified in several viruses that are also present in animal tissue, such as muscle or nervous tissue (eg, myelin basic protein). The antibodies to these epitopes might contribute to immune-mediated tissue damage during the course of viral infection, but their pathogenic role, if any, in initiating and potentiating autoimmune disease remains uncertain.
Persistent Infection and Chronic Damage to Tissues and Organs
Persistent infections of one type or another are produced by a wide range of viruses, and are common in veterinary medicine. Apart from enteric and respiratory viruses that cause transient infections that remain localized to their respective target organs, most other categories of viral infections include examples of persistent infection. Foot-and-mouth disease, for example, usually is an acute, self-limiting infection, but a carrier state of uncertain epidemiological relevance occurs in which virus persists in the oropharynx of a very few convalescent animals. In other instances, such as those associated with immunodeficiency viral infections, persistent viral infections lead to chronic diseases, even when the acute manifestations of infection have been trivial or subclinical. Finally, persistent infections can lead to continuing tissue injury, often with an immune-mediated basis. Persistent viral infections are important for several reasons. For example, they may be reactivated and cause recrudescent episodes of disease in the individual host, or they may lead to immune-mediated disease or to neoplasia. Persistent infection may allow survival of a particular virus in individual animals and herds, even after vaccination. Similarly, persistent infections may be of epidemiologic importance—the source of contagion in long-distance virus transport and in reintroduction after elimination of virus from a given herd, flock, region, or country. Persistent infections are manifest in several ways. There are persistent infections in which virus is demonstrable continuously, whether or not there is ongoing disease. Disease may develop late, often with an immunological or neoplastic basis. In other instances, disease is not manifest in persistently infected animals; for example, in the deer mouse (Peromyscus maniculatus), the reservoir rodent host of Sin Nombre virus, and the etiologic agent of hantavirus pulmonary syndrome in humans, virus is shed in urine, saliva, and feces probably for the life of the animal, even in the face of neutralizing antibody. A striking proportion of persistent infections involve the CNS. Restrictions in antigen presentation by neurons and glia and the activity of regulatory T cells combine to tightly regulate immune responses in the CNS. This regulation is important in order to assure that immune and inflammatory responses do not disrupt the highly specialized functions of terminally differentiated neurons and myelin producing cells. Myelin also contains unique antigens capable of eliciting autoimmune reactions, further emphasizing the importance of regulating immune responses in the CNS. This environment poses an excellent opportunity for a virus to avoid immune surveillance, and neurons and glia often exhibit limited permissiveness to virus gene expression that favors a noncytopathic persistent infection.
Latent infections are a form of persistence in which infectious virus is not demonstrable except when reactivation occurs. For example, in infectious pustular vulvovaginitis, the sexually transmitted disease caused in cattle by bovine herpesvirus 1, virus usually cannot be isolated from the latently infected carrier cow except when there are recrudescent lesions. Viral latency may be maintained by restricted expression of genes that have the capacity to kill the cell. During latency, herpesviruses express only a few genes that are necessary in the maintenance of latency, notably so-called latency-associated transcripts. During reactivation, which is often stimulated by immunosuppression and/or by the action of a cytokine or hormone, the whole viral genome is transcribed again. This strategy protects the virus during its latent state from all host immune actions that would normally result in virus clearance. The dynamic nature of viruscell or virustissue interactions gives rise to a spectrum of clinical manifestations of disease associated with viral persistence (Fig. 3.14). Slow infections is a clinical term used to describe a slowly progressive disease, where the initiation of infection is subclinical, and evidence of disease builds slowly as the virus persists. Persistence is associated with a progressive increase in viral burden and antigen expression and the associated inflammatory and immune responses that are the basis for disease. An example of a slow virus infection is ovine progressive pneumonia caused by retrovirus infection (see Chapter 14: Retroviridae). In chronic diseases, there may be evidence of the initiation of infection (ie, the acute clinical episode) followed by the clinical manifestations of persistence in which disease progresses more rapidly following an incubation period. Canine distemper virus infection in the CNS can be manifest as a chronic disease following acute multisystemic infection, although the initial infection may go unrecognized and the incubation period for the appearance of clinical neurological disease may be prolonged as in a slow virus infection. Viruses that undergo latency with episodes of periodic reactivation may similarly be manifest as chronic diseases. These examples highlight the limitations of using clinical terminology to describe virus infection of a host, where the presence or absence of the virus or the different types of virustissue interactions are considered separately. To further illustrate this latter point are examples where acute infections have late clinical manifestations in which continuing replication of the causative virus is not involved in the progression of the disease. For example, in the cerebellar hypoplasia syndrome that occurs in young cats as a result of fetal infection with feline panleukopenia virus, virus cannot be isolated at the time neurologic damage is diagnosed. In fact, because of this, the cerebellar syndrome was for many years considered to be an inherited malformation. Further, some persistent infections possess features of more than one of these categories. For example, all retrovirus infections are persistent and most exhibit features of latency, but the diseases they cause may be delayed following infection or only manifest as slowly progressive diseases. Individual viruses employ a remarkable variety of strategies for successful evasion of host immune and inflammatory responses in vivo. These mechanisms include noncytocidal infections without expression of immunogenic proteins, replication in cells of the immune system or subversion of host innate and adaptive immunity (see Chapter 4: Antiviral Immunity and Virus Vaccines), and infection of nonpermissive, resting, or undifferentiated cells. Some viruses have evolved strategies for evading neutralization by the antibody they elicit. Ebola virus, for example, uses an “immune decoy” to evade neutralizing antibody—specifically, a secreted viral protein that binds circulating antibody. The surface glycoproteins of filoviruses, arenaviruses, bunyaviruses (eg, Rift Valley fever virus), and some arteriviruses (eg, porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus) are heavily glycosylated, which may serve to mask the neutralizing epitopes contained in these proteins. Antigenic drift is especially characteristic of persistent RNA viral infection, particularly for lentiviruses (eg, equine infectious anemia virus). During persistent infection, sequential antigenic variants are produced, with each successive variant sufficiently different to evade the immune response raised against the preceding variant. In equine infectious anemia, clinical signs occur in periodic cycles, with each cycle being initiated by the emergence of a new viral variant. In addition to providing a mechanism for escape from immune elimination, each new variant may be more virulent than its predecessor, and this may directly affect the severity and progression of the disease. The integration of retroviral proviral DNA into the genome of the host germ-line cells assures indefinite maintenance from one generation to the next; such proviral DNA can also lead to induction of tumors (oncogenesis).
VIRUS-INDUCED NEOPLASIA
Neoplasms arise as a consequence of the dysregulated growth of cells derived from a few or a single, genetically altered progenitor cell(s). Thus, although neoplasms are often composed of several cell types, they are considered to originate from an oligoclonal or monoclonal outgrowth of a single cell. The genetic changes that are ultimately responsible for neoplasia may be caused by naturally occurring mutations, chemical or physical agents or infectious agents including viruses, but all involve certain common cellular pathways. The discoveries of the viral etiology of avian leukemia by Ellerman and Bang and of avian sarcoma by Rous, in 1908 and 1911, respectively, were long regarded as curiosities unlikely to be of any fundamental significance. However, study of these avian viruses and related retroviruses of mice has increased our overall understanding of neoplasia greatly, and since the 1950s there has been a steady stream of discoveries clearly incriminating other viruses in a variety of benign and malignant neoplasms of numerous species of mammals, birds, amphibians, reptiles, and fish. Many avian retroviruses are major pathogens of poultry, and several DNA viruses have been determined to be responsible for cancers in humans and animals. Any discussion of virus-induced neoplasia requires that a few commonly used terms are defined: a neoplasm is a new growth (syn. tumor) which can be benign or malignant; neoplasia is the process that leads to the formation of neoplasms (syn. carcinogenesis); oncology is the study of neoplasia and neoplasms; a benign neoplasm is a growth produced by abnormal cell proliferation that remains localized and does not invade adjacent tissue; in contrast, a malignant neoplasm (syn. cancer) is locally invasive and may also be spread to other parts of the body (metastasis). Carcinomas are cancers of epithelial cell origin, whereas sarcomas are cancers that arise from cells of mesenchymal origin. Solid neoplasms of lymphocytes are designated lymphosarcoma, malignant lymphoma, or lymphoma, whereas leukemias are cancers of hemopoietic origin characterized by circulation of cancerous cells.
The Cellular Basis of Neoplasia
Neoplasia is the result of nonlethal genetic injury, as may be acquired by chemical or physical damage, or from viral infections. Some cancers, however, arise randomly through the accumulation of spontaneous genetic mutations. A neoplasm results from the clonal expansion of cells that have suffered genetic damage, typically in one of four types of normal regulatory genes: (1) protooncogenes, which are cellular genes that regulate growth and differentiation; (2) tumor suppressor genes that inhibit growth, typically by regulating the cell cycle; (3) genes that regulate apoptosis (programmed cell death); (4) genes that mediate DNA repair. Carcinogenesis involves a multistep progression resulting from the cumulative effects of multiple mutations. Once developed, neoplasms are: (1) self-sufficient, in that they have the capacity to proliferate without external stimuli; for example, as the result of unregulated oncogene activation; (2) insensitive to normal regulatory signals that would limit their growth, such as transforming growth factor and the cyclin-dependent kinases that normally regulate orderly progression of cells through the various phases of the cell cycle; (3) resistant to apoptosis because of either the activation of antiapoptotic molecules or the inhibition of mediators of apoptosis such as p53; (4) limitless potential for replication. Cancers also may have the ability to invade and spread to distant tissues (metastasis), and neoplasms typically promote the proliferation of new blood vessels that support their growth. Neoplasia, regardless of cause, is the result of unregulated cellular proliferation. In the normal sequence of events during cellular proliferation, a growth factor binds to its specific cellular receptor, leading to signal transduction that ultimately results in nuclear transcription, which in turn leads to the cell entering and progressing through the cell cycle until it divides. Proto-oncogenes are normal cellular genes that encode proteins that function in normal cellular growth and differentiation; they include (1) growth factors; (2) growth factor receptors; (3) intracellular signal transducers; (4) nuclear transcription factors; (5) cell cycle control proteins. Oncogenes are derived by mutation of their normal cellular proto-oncogene counterparts, and the expression of oncogenes results in production of oncoproteins that mediate autonomous (unregulated) growth of neoplastic cells. The development of cancer (malignant neoplasia) is a protracted, multistep process that reflects the accumulation of multiple mutations. Potentially neoplastic cells must bypass apoptosis (programmed death), circumvent the need for growth signals from other cells, escape from immunologic surveillance, organize their own blood supply, and possibly metastasize. Thus, tumors other than those induced by rapidly transforming retroviruses like Rous sarcoma virus generally do not arise as the result of a single event, but by a series of steps leading to progressively greater loss of regulation of cell division. Viruses are classified as tumor viruses if part of the viral genome is present in tumors, with expression within the tumor of some viral genes. In vitro, infection of cells with tumor viruses leads to transformation caused by specific viral genes. Infection of experimental animals leads to tumor formation that is preventable by vaccination, although this experiment cannot be performed with most human viruses because they do not infect rodents. Oncogenic DNA viruses (eg, papillomaviruses, polyomaviruses, herpesviruses) and RNA viruses (retroviruses) have been identified in both animals and humans. DNA viruses can cause neoplasia by inhibiting tumor suppressor genes whereas RNA viruses typically activate protooncogenes. Cells transformed by nondefective retroviruses also express the full range of viral proteins, and new virions bud from their membranes. In contrast, transformation by DNA viruses usually occurs in cells undergoing nonproductive infection in which viral DNA is integrated into the cellular DNA of the transformed cells or, in the case of papillomaviruses, polyomaviruses and herpesviruses, in which the viral DNA remains episomal. Certain virus-specific antigens are demonstrable in transformed cells.
Oncogenic RNA Viruses
Retrovirus-Induced Neoplasia
Retroviruses are a significant cause of neoplasia in many species of animals, including cattle, cats, nonhuman primates, mice, and birds, among others. Their pathogenesis is linked to their propensity to integrate within the genome of host cells, thereby being infectious mutagens. The consequences of such integration are largely innocuous and clinically silent, and only seldom result in oncogenesis. As described in Chapter 14, Retroviridae, retroviruses can be biologically divided into exogenous (horizontally transmissible) agents, or endogenous. Retroviruses can be either replication-competent or replication-defective. Oncogenic retroviruses are classified as acute transforming or chronic transforming retroviruses. These two major types of transforming retroviruses induce neoplasia in significantly different ways.
Acute Transforming Retroviruses
Acute transforming retroviruses infect mice and birds and are directly oncogenic by carrying an additional viral oncogene, v-onc, and are classified as “transducing” retroviruses. The retroviral v-onc originates from a host c-onc gene, where the transforming activity of the v-onc is accentuated by mutation. These mutations reflect the high error rate of the viral reverse transcriptase. Other viral oncogenes may induce cellular transformation simply by overexpression (from the viral promoter), independent of any mutations. These acquired genes are components of the cell signaling networks and the strongly promoted production of the viral oncoprotein will readily exceed that of the normal cellular oncoprotein. The result can be uncontrolled cell growth. Because c-onc genes are the precursors of v-onc genes, c-onc genes are also called “proto-oncogenes.” Wherever acute transforming retroviruses integrate in the host genome, it is the v-onc that is directly responsible for the rapid malignant change that occurs in cells infected with these viruses. Over 60 different v-onc genes have been identified, and retroviruses have been instrumental in identifying their cellular homologues. The v-onc is usually incorporated into the viral genomic RNA, replacing a portion of one or more normal viral genes. Because such viruses have lost some of their viral genetic sequences, they are usually incapable of replication, and are therefore termed “defective” retroviruses. An exception is Rous sarcoma virus, in that its genome contains a viral oncogene (v-src) in addition to its full complement of functioning viral genes (gag, pol, and env); thus Rous sarcoma virus is both replication-competent and an acute transforming virus. Rous sarcoma virus is one of the most rapidly acting carcinogens known, transforming cultured cells in a day or so and causing neoplasia and death in chickens in as little as 2 weeks after infection. Defective retroviruses circumvent their defective replicative ability by utilizing nondefective “helper” retroviruses for formation of infectious virions. Replication of the defective virus is thus said to be “rescued” by helper viruses that provide the missing function (eg, an environmentally stable envelope). Although v-onc genes often compromise retrovirus replication, v-onc genes may be acquired over time by integrated proviruses, most likely because of the effects on cell proliferation that would amplify v-onc containing cells. Cell proliferation also favors replication of helper virus that can rescue the v-onc containing defective virus, thereby facilitating direct viral dissemination of v-onc within a host. The various v-onc genes and the proteins they encode can be assigned to major classes: growth factors (such as v-sis); growth factor receptors and hormone receptors (such as v-erbB); intracellular signal transducers (such as v-ras); and nuclear transcription factors (such as v-jun). The oncoprotein products of the various retroviral v-onc genes act in many different ways to affect cell growth, division, differentiation, and homeostasis:
1. v-onc genes usually contain only that part of their corresponding c-onc gene that is transcribed into messenger RNA—in most instances they lack the introns that are so characteristic of eukaryotic genes.
2. v-onc genes are separated from the cellular context that normally controls gene expression, including the normal promoters and other sequences that regulate conc gene expression.
3. v-onc genes are under the control of the viral long terminal repeats (LTRs), which not only are strong promoters but also are influenced by cellular regulatory factors. For some retrovirus v-onc genes, such as myc and mos, the presence of viral LTRs is all that is needed for tumor induction.
4. v-onc genes may undergo mutations (deletions and rearrangements) that alter the structure of their protein products; such changes can interfere with normal proteinprotein interactions, leading to escape from normal regulation.
5. v-onc genes may be joined to other viral genes in such a way that their functions are modified. For example, in Abelson murine leukemia virus the v-abl gene is expressed as a fusion protein with a gag protein; this arrangement directs the fusion protein to the plasma membrane where the Abl protein functions. In feline leukemia virus, the v-onc gene fms is also expressed as a fusion protein with a gag protein, thus allowing the insertion of the Fms oncoprotein in the plasma membrane.
Infection with acute transforming retroviruses may lead to transformation of every infected cell and therefore to very rapid tumor development (sometimes within days).
Chronic Transforming Retroviruses Chronic transforming retroviruses induce neoplasia through integration into the genome of somatic cells. Recent research suggests that the selectivity of integration sites is specific for individual retrovirus species, and thereby contribute to pathogenicity. Chronic transforming retroviruses are classified as cis- or trans-acting. “Cis-acting” retroviruses (eg, avian leukosis viruses) transform cells by becoming integrated in the host-cell DNA close to a cell growth regulating gene, and thus usurping normal cellular regulation of this gene. These cell growth regulating host genes are termed “proto-oncogenes,” or cellular oncogenes (c-onc). Despite the terminology implying that they are oncogenic, c-onc genes are host genes that encode important cell signaling products that regulate normal cell proliferation and quiescence. The presence of an integrated provirus, with its strong promoter and enhancer elements, upstream from a c-onc gene may amplify the expression of the c-onc gene greatly. This is the likely mechanism whereby the weakly oncogenic endogenous avian leukosis viruses produce neoplasia. When avian leukosis viruses cause malignant neoplasia, the viral genome has generally been integrated at a particular location, immediately upstream from a host c-onc gene. Integrated avian leukosis provirus increases the synthesis of the normal c-myc oncogene product 30- to 100-fold. Experimentally, only the viral LTRs need be integrated to cause this effect; furthermore, by this mechanism c-myc may also be expressed in cells in which it is not normally expressed or is normally expressed at much lower levels. Infection with cis-acting retroviruses results in transformation of single cells (monoclonal tumor) and slow tumor formation over months. “Trans-activating” retroviruses express viral proteins that act as oncogenes. The retroviruses that cause nasal carcinomas and pulmonary adenocarcinomas (Jaagsiekte) in sheep infect epithelial cells, and transformation is related to expression of the viral env gene. Bovine leukemia virus is an exogenous retrovirus that causes chronic leukosis and B cell lymphoma. Its Tax protein functions as a transactivator of host genes. Both the ovine retrovirus Env and the Tax proteins of bovine leukemia virus stimulate continuous cell division of infected cells, which is thought to result in an increased number of mutations and subsequently cellular transformation. Infection with transacting retroviruses leads to oligoclonal tumors which develop over months to years.
Oncogenic DNA Viruses
Apart from retroviruses, the most important oncogenic viruses in animals are DNA viruses (papillomaviruses, polyomaviruses, herpesviruses; see also Table 3.4). DNA tumor viruses interact with cells in one of two ways: (1) productive infection, in which the virus completes its replication cycle, resulting in cell lysis or (2) nonproductive infection, in which the virus transforms the cell without completing its replication cycle. During such nonproductive infection, the viral genome or a truncated version of it is integrated into the cellular DNA or the complete genome persists as an autonomously replicating plasmid (episome). The genome continues to express early genes. The molecular basis of oncogenesis by DNA viruses is best understood for polyomaviruses, papillomaviruses, and adenoviruses, all of which contain genes that behave as oncogenes, including tumor suppressor genes. These oncogenes appear to act by mechanisms similar to those described for retrovirus oncogenes: they act primarily in the nucleus, where they alter patterns of gene expression and regulation of cell growth. The relevant proteins have a dual role in both virus replication and cell transformation. With a few possible exceptions, the oncogenes of DNA viruses have no homologue or direct ancestors (conc genes) among cellular genes of the host.
Oncogenic Papillomaviruses
Papillomaviruses produce papillomas (warts) on the skin and mucous membranes of most animal species (see Chapter 11: Papillomaviridae and Polyomaviridae). Papillomas are hyperplastic epithelial outgrowths that generally regress spontaneously. Occasionally, however, infections by some papillomavirus types may cause malignant cellular transformation, resulting in the development of cancer. Papillomaviruses are known to cause oropharyngeal and cervical squamous cell carcinomas in people. In animals, papillomaviruses are also thought to cause sarcoids in horses, and have been associated with some squamous cell carcinomas in horses, cats and dogs. In warts, the papillomavirus DNA remains episomal, meaning it is not integrated into the host-cell DNA and persists as an autonomously replicating episome. In contrast, in human papillomavirus-induced neoplasms the viral DNA is integrated into that of the host. As the pattern of integration is clonal within cancers, each cancer cell carries at least one, and often many incomplete copies of the viral genome. The site of virus integration is random, and there is no consistent association with cellular proto-oncogenes. For some papillomaviruses, integration disrupts one of the early genes, E2, which is a viral repressor. Other viral genes may also be deleted, but the viral oncogenes (eg, E6 and E7) remain intact. These oncogenes alter normal cell growth and division and the overexpression of E6 and E7 is considered a critical step in malignant transformation by a human papillomavirus. It is to be stressed that the development of warts is a normal part of viral replication cycle of some papillomavirus types. However, the integration of DNA into a cell is accidental and prevents replication of the papillomavirus and only a very small proportion of papillomavirus infections result in cancer development. However, bovine papillomavirus type 1 is thought to cause equine sarcoids predominantly through changes in cell proliferation that are mediated by the E5 oncoprotein. In contrast to human papillomavirus-induced cancers, viral integration appears to be uncommon within papillomavirus-associated cancers in animals.
Oncogenic Hepadnaviruses
Mammalian, but not avian, hepadnaviruses are associated strongly with naturally occurring hepatocellular carcinomas in their natural hosts. Woodchucks that are chronically infected with woodchuck hepatitis virus almost inevitably develop hepatocellular carcinoma, even in the absence of other carcinogenic factors. Oncogenesis induced by mammalian hepadnaviruses is a multifactorial process, and there are differences in the cellular mechanisms responsible for carcinogenesis associated with different viruses. Whereas ground squirrel and woodchuck hepatitis viruses activate cellular oncogenes, the mode of action of human hepatitis B virus is uncertain, as it apparently has no consistent site of integration or oncogene association. The hepatocellular regeneration accompanying cirrhosis of the liver also promotes the development of neoplasia in hepatitis virus-infected humans, but there is no cirrhosis in the animal models. The likelihood of hepadnavirus-associated carcinoma is greatest in animals (and humans) infected at birth.
Oncogenic Herpesviruses
Marek’s disease virus of chickens (gallid herpesvirus 2) transforms T lymphocytes, causing them to proliferate to produce a generalized polyclonal T lymphocyte neoplasm. The disease is preventable by vaccination with liveattenuated virus vaccines that lack the retrovirus v-onc genes that are present in Marek’s disease virus. The best characterized oncogene is the Meq protein, which inhibits tumor suppressor genes and stimulates expression of proteins important for cell growth (IL-2, Bcl-2, CD30). It also binds to the promoter of and stimulates the expression of micro RNA21, which subsequently causes expression of metalloproteinases required for tissue invasion by tumor cells.
Oncogenic Poxviruses
Although some poxviruses are regularly associated with the development of benign tumor-like lesions (see Chapter 7: Poxviridae), there is no evidence that these ever become malignant, nor is there evidence that poxvirus DNA is ever integrated into cellular DNA. A very early viral protein produced in poxvirus-infected cells displays homology with epidermal growth factor and is probably responsible for the epithelial hyperplasia characteristic of many poxvirus infections. For some poxviruses (eg, fowlpox, orf, and rabbit fibroma viruses), epithelial hyperplasia is a dominant clinical manifestation and may be a consequence of a more potent form of the poxvirus epidermal growth factor homologue.
Oncogenic in Experimental Systems: Polyomaviruses and Adenoviruses
During the 1960s and 1970s, two members of the family Polyomaviridae, murine polyomavirus and simian virus 40 (SV40), as well as certain human adenoviruses (types 12, 18, and 31) were shown to induce malignant neoplasms following their inoculation into baby hamsters and other rodents. With the exception of murine polyomavirus, none of these viruses induces cancer under natural conditions in its natural host, rather they transform cultured cells of certain other species and provide experimental models for analysis of the molecular events in cell transformation. More recently, polyomaviruses have been incriminated as the cause of cancers in both humans and animals (see Chapter 11: Papillomaviridae and Polyomaviridae). Polyomavirus- or adenovirus-transformed cells do not produce virus. Viral DNA is integrated at several sites in the chromosomes of the cell. Most of the integrated viral genomes are complete in the case of the polyomaviruses, but defective in the case of the adenoviruses. Only certain early viral genes are transcribed, albeit at an unusually high rate. By analogy with retrovirus genes, they are now called oncogenes. Their products, demonstrable by immunofluorescence, used to be known as tumor (T) antigens. A great deal is now known about the role of these proteins in transformation. Virus can be rescued from polyomavirus-transformed cells—that is, virus can be induced to replicate by irradiation, treatment with certain mutagenic chemicals, or cocultivation with certain types of permissive cells. This cannot be done with adenovirustransformed cells, as the integrated adenovirus DNA contains substantial deletions.
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