Viral infection is not synonymous with disease, as many viral infections are subclinical (syn., asymptomatic, inapparent), whereas others result in disease of varying severity that is typically accompanied by characteristic clinical signs in the affected host (Fig. 3.1).
Amongst many other potentially contributing factors, the outcome of the virus host encounter is essentially the product of the virulence of the infecting virus on the one hand and the susceptibility of the host on the other.
The term virulence is used as a quantitative or relative measure of the pathogenicity of the infecting virus—that is, a virus is said to be either pathogenic or nonpathogenic, but its virulence is stated in relative terms (“virus A is more virulent than virus B” or “virus strain A is more virulent in animal species Y than species Z”).
The terms pathogenicity and virulence refer to the capacity of a virus to cause disease in its host, and are unrelated to the infectivity or transmissibility (contagiousness) of the virus. For viruses to cause disease they must first infect their host, spread within the host, and damage target tissues.
To ensure their propagation, viruses must then be transmitted to other susceptible individuals—that is, they must be shed within secretions or excretions into the environment, be taken up by another host or a vector, or be passed congenitally from mother to offspring. Viruses have developed a remarkable variety of strategies to ensure their own survival. Similarly, individual viruses cause disease through a considerable variety of distinct pathogenic mechanisms.
INTERPLAY OF VIRAL VIRULENCE AND HOST RESISTANCE, OR SUSCEPTIBILITY FACTORS IN MANIFESTATION OF VIRAL DISEASES
Viruses differ greatly in their virulence, but even in a population infected by a particular virus strain there are usually striking differences in the outcome of infection between individual animals.
Similarly, there is much variation amongst viruses of the same species and the determinants of viral virulence are often multigenic, meaning that several viral genes contribute to the virulence of individual viruses.
The determinants of host resistance/susceptibility are usually multifactorial, and include not only a variety of host factors but environmental ones as well. The advent and application of molecular technologies has facilitated mapping of virulence determinants in the genome of many viruses (eg, by whole-genomic sequencing of virus strains, and manipulation of molecular clones), as well as resistance/susceptibility determinants in the genome of experimental animals.
Virus strain differences may be quantitative, involving the rate and yield of virus replication, lethal dose, infectious dose, the number of cells infected in a given organ, or they may be qualitative, involving organ or tissue tropism, extent of host-cell damage, mode and efficacy of spread in the body, and character of the disease they induce.
Assessment of Viral Virulence
There is wide variation in the virulence of viruses, ranging from those that almost always cause inapparent infections, to those that usually cause disease, to those that usually cause death.
Meaningful comparison of the virulence of viruses requires that factors such as the infecting dose of the virus and the age, sex, and condition of the host animals and their immune status be equal; however, these conditions are never met in nature, where heterogeneous, outbred animal populations are the rule and the dynamics of exposure and viral infection are incredibly varied.
Hence, subjective and vague terminology may be used to describe the virulence of particular viruses in domestic and wild animals. Precise measures of virulence are usually derived only from assays in inbred animals such as mice.
Of course, such assays are only feasible for those viruses that grow in mice, and care must always be exercised in extrapolating data from laboratory mice to the host species of interest. The virulence of a particular strain of virus administered in a particular dose, by a particular route, to a particular age and strain of laboratory animal may be assessed by determining its ability to cause disease, death, specific clinical signs, or lesions. The dose of the virus required to cause death in 50% of animals (lethal dose 50, LD50) has been a commonly used measure of virulence, but is now passing out of favor in the research arena for ethical reasons.
For example, in the susceptible BALB/c strain of mouse, the LD50 of a virulent strain of ectromelia virus is 5 virions, as compared with 5000 for a moderately attenuated strain and about 1 million for a highly attenuated strain. Viral virulence can also be measured in experimental animals by determining the ratio of the dose of a particular strain of virus that causes infection in 50% of individuals (infectious dose 50, ID50) to the dose that kills 50% of individuals (the ID50:LD50 ratio).
Thus, the ID50 of a virulent strain of ectromelia virus in BALB/c mice is 2 virions and the LD50 about 5 virions, whereas for resistant C57BL/6 mice the ID50 is the same but the LD50 is 1 million virions. The severity of an infection, therefore, depends on the interplay between the virulence of the virus and the resistance of the host. Viral virulence also can be estimated through assessment of the severity, location, and distribution of gross, histologic, and ultrastructural lesions in affected animals.
Determinants of Viral Virulence
The advent of molecular biology has facilitated determination of the genetic basis of virulence of many viruses, along with other important aspects of their replication. Specifically, the role of potential determinants of virulence identified by genetic sequence comparison of viruses of defined virulence can be confirmed unequivocally by manipulation of molecular clones of the virus in question.
This “reverse genetics” strategy utilizing molecular (infectious) clones was first widely employed using complementary DNA (cDNA) copies of the entire genome of simple positive-strand RNA viruses such as alphaviruses and picornaviruses, where RNA transcribed from the full-length cDNA copies (clones) of the genomes of such viruses is itself capable of initiating the viral replication cycle following transfection into cells. The genomic RNA of negative-sense RNA viruses such as rhabdoviruses is not in and of itself infectious, but infectious virus can be recovered from cDNA clones if viral proteins supporting genome replication are also produced in cells transfected with genome-length RNA transcripts. Even the considerable logistical challenges posed by RNA viruses with segmented genomes (such as influenza viruses, bunyaviruses, arenaviruses, and reoviruses) have been overcome, and molecular clones of these viruses are now used for reverse genetic manipulation.
It is also possible to specifically manipulate the genomes of even the very large DNA viruses as artificial chromosomes. Of necessity, most experimental work has been carried out in inbred laboratory animals, although molecular clones of a substantial number of pathogenic animal viruses have now been evaluated in their respective natural animal hosts. It is apparent from these reverse genetic studies that several viral genes can contribute to the virulence of individual viruses, as described under each virus family in Part II of this book. Viruses exhibit host and tissue specificity (tropism), usually more than is appreciated clinically. Mechanistically, the organ or tissue tropism of the virus is an expression of all the steps required for successful infection, from the interaction of virus attachment molecules and their cellular receptors to virus assembly and release (see Chapter 2: Virus Replication).
Organ and tissue tropisms also involve all stages in the course of infection in the whole host animal, from the site of entry, to the major target organs responsible for the clinical signs, to the site involved in virus release and shedding. Caution should be exercised in attributing characteristics of viral epidemics solely to the virulence of the causative virus, as there typically is considerable variation in the response of individual infected animals, both within and between animal species.
For example, during the epizootic of West Nile virus infection that began in North America in 1999, approximately 10% of infected horses developed neurological disease (encephalomyelitis) and, of these, some 3035% died. Neuroinvasive disease was even less common in humans infected with this same strain of West Nile virus, whereas infected corvids (crows and their relatives) almost uniformly developed disseminated, rapidly fatal infections.
Determinants of Host Resistance/ Susceptibility
As just described for West Nile virus, genetic differences in host resistance/susceptibility to viral infections are most obvious when different animal species are compared. Viral infections tend to be less pathogenic in their natural host species than in exotic or introduced species. For instance, myxoma virus produces a small benign fibroma in its natural host, which are wild rabbits of the Americas (Sylvilagus spp.), but the same virus almost invariably causes a fatal generalized infection in the European rabbit, Oryctolagus cuniculus.
Likewise, zoonotic (transmitted from animal to human) infections caused by arenaviruses, filoviruses, paramyxoviruses, coronaviruses, and many arboviruses are severe in humans but mild or subclinical in their reservoir animal hosts. The innate and adaptive immune responses to particular viral infections differ greatly from one individual to another (see Chapter 4: Antiviral Immunity and Virus Vaccines). Studies with inbred strains of mice have confirmed that susceptibility to specific viruses may be associated with particular major histocompatibility (MHC) antigen haplotypes, presumably because of their central role in directing the nature of the adaptive immune response generated to the infecting virus. Similarly, studies with genetically modified mice have unequivocally confirmed the critical role of innate immune responses, especially those associated with the interferon system, in conferring antiviral resistance and protection. Expression of critical receptors on target cells is a fundamental determinant of host resistance/susceptibility to a particular virus.
The more conserved or ubiquitous the receptor, the wider the host range of the virus that exploits it; for example, rabies virus, which uses sialylated gangliosides in addition to the acetylcholine receptor, has a very wide host range, but infection is restricted narrowly to a few host cell types, including myocytes, neurons, and salivary gland epithelium. Changes in viral attachment proteins can lead to the emergence of variant viruses with different tropism and disease potential. For example, porcine respiratory coronavirus arose from transmissible gastroenteritis virus, which is strictly an enteric pathogen, through a substantial deletion in the gene encoding the viral spike protein that mediates virus attachment. This change affected the tropism of the virus as well as its transmissibility.
Physiologic Factors Affecting Host Resistance/Susceptibility
In addition to innate and adaptive immune responses, a considerable variety of physiologic factors affect host resistance/susceptibility to individual viral diseases, including age, nutritional status, levels of certain hormones, and cell differentiation. Viral infections tend to be most serious at both ends of life—in the very young and the very old. Rapid physiologic changes occur during the immediate postpartum period and resistance to the most severe manifestations of many intestinal and respiratory infections builds quickly in the neonate. Maturation of the immune system is responsible for much of this enhanced, age-related resistance, but physiologic changes also contribute. Malnutrition can also potentially impair immune responsiveness in adults, but it is often difficult to distinguish adverse nutritional effects from other factors found in animals living in very adverse environments. Certain infections, particularly herpesvirus infections, can be reactivated during pregnancy, leading to abortion or perinatal infection of the progeny of infected dams. The fetus itself is uniquely susceptible to a number of different viral infections, reflecting immaturity of the immune system, immaturity of biological barriers (eg, the bloodbrain barrier) and increased permissiveness of rapidly dividing cell populations, the latter being abundant in developing tissues. Cellular differentiation and the stage of the cell cycle may affect susceptibility to infection with specific viruses. For example, parvoviruses replicate only in cells that are in the late S phase of the cell cycle, so the rapidly dividing cells of bone marrow, intestinal epithelium, and the developing fetus are vulnerable. The rapidly dividing, often migratory cell populations that occur during embryogenesis in the developing fetus are exquisitely susceptible to infection and injury by a number of viruses, notably several highly teratogenic viruses that infect the developing central nervous system (CNS). Almost all viral infections are accompanied by fever. In classic studies of myxoma virus infection in rabbits, it was shown that increasing body temperature increased protection against disease, whereas decreasing temperature increased the severity of infection. Blocking the development of fever with drugs (eg, salicylates) increased mortality. Similar results have been obtained with ectromelia and coxsackievirus infections in mice. In contrast, fever does not accompany viral infection in certain poikilotherms (eg, fish), in which this response is probably of no or lesser selective advantage. The immunosuppressive effects of increased concentrations of corticosteroids, whether endogenous or exogenous in origin, can reactivate latent viral infections or exacerbate active mild or subclinical viral infections, such as those caused by herpesviruses. This mechanism probably contributes to the increased incidence of severe viral infections that occurs in settings in which animals are stressed as a result of transport and/or introduction into crowded environments, such as animal shelters and feedlots. Products of host inflammatory and innate immune responses also probably contribute to the transient immunosuppression and other general signs that can accompany viral infections.
MECHANISMS OF VIRAL INFECTION AND VIRUS DISSEMINATION
At the level of the cell, infection by viruses (see Chapters 1 and 2: The Nature of Viruses and Virus Replication) is quite different from that caused by bacteria and other microorganisms, whereas at the level of the whole animal and animal populations there are more similarities than differences.
Like other microorganisms, viruses must gain entry into their host’s body before they can exert their pathogenic effects; entry of virus into the host can occur through any of a variety of potential routes, depending on the properties of the individual virus (Table 3.1).
Routes of Virus Entry
Viruses are obligate intracellular parasites that are transmitted as inert particles.
To infect its host, a virus must first attach to and infect cells at one of the body surfaces, either the integument or a mucosal surface.
The skin that covers the animal body externally has a relatively impermeable outer layer of keratin, and initiation of infection may require that this barrier be compromised or even bypassed via a wound such as a needle stick, insect or animal bite.
Barriers to the initiation of infection on mucosal surfaces are much less formidable, specifically on the mucosal epithelial lining of the respiratory, gastrointestinal, and urogenital tracts and the nonkeratinized epithelial lining of the conjunctiva and cornea of the eyes.
In animals without significant areas of keratinized epithelium (eg, fish), the skin and gills serve as an extensive mucosal surface that is the initial site of infection with many viruses. Virus replication may subsequently be limited to the body surface through which the virus entered or the virus may be disseminated to replicate in multiple tissues, with subsequent shedding from body surfaces that are either the same or different from the route of entry (Fig. 3.2).
FIGURE 3.2 Relationship between body surfaces that are routes of viral entry, the nature of viral spread, and body surfaces that shed progeny virus. Entry into the respiratory tract is shown here as an example. Local spread from the route of entry restricts viral shedding to respiratory secretions, as in the case of canine parainfluenza virus infection. Viremic spread from the route of entry results in shedding from multiple mucosal surfaces. In the case of canine distemper virus infection, this includes respiratory mucosa (the route of entry) and those of other organ systems, such as the urinary tract.
Entry via the Respiratory Tract
The mucosal surfaces of the respiratory tract are lined by epithelial cells that can potentially support the replication of viruses, so defenses are necessary to minimize the risk of infection. The respiratory tract from the nasal passages to the distal airways in the lungs is protected by the “mucociliary blanket,” which consists of a layer of mucus produced by goblet cells that is kept in continuous flow by the coordinated beating of cilia on the luminal surface of the epithelial cells that line the nasal mucosa and airways.
Inhaled virions can be trapped in the viscous mucus layer and then carried by ciliary action from the nasal cavity and airways to the pharynx, where they are then swallowed or coughed out. The distance to which inhaled particles penetrate into the respiratory tract is inversely related to their size, so that larger particles (greater than 10 μm in diameter) are trapped on the mucociliary blanket lining the nasal cavity and airways and small particles (less than 5 μm in diameter) can be inhaled directly into the airspaces of the lungs (alveoli), where they are ingested by resident alveolar macrophages.
The respiratory system is also protected by innate and adaptive immune mechanisms that operate at all mucosal surfaces (see Chapter 4: Antiviral Immunity and Virus Vaccines), including specialized lymphoid aggregates that occur throughout the respiratory tree [eg, nasal-associated lymphoid tissue (NALT) and tonsils, and bronchusassociated lymphoid tissue (BALT)]. Despite its protective mechanisms, however, the respiratory tract is perhaps the most common portal of virus entry into the body.
Environmental factors may enhance infection by compromising defense mechanisms. For example, exposure to ammonia vapor causes ciliary stasis, and serous effusions associated with inflammation can dilute the viscosity of the mucus layer, both of which can enhance a virus’ ability to attach to specific receptors on epithelial cells within the mucosa. After invasion, some viruses remain localized to the respiratory system or spread from cell to cell to invade other tissues, whereas many others become widely disseminated via lymphatics and/or the bloodstream.
Entry via the Gastrointestinal Tract
A substantial number of viruses (enteric viruses) are spread to susceptible hosts by ingestion of viruscontaminated food or drink. The mucosal lining of the oral cavity and esophagus (and forestomachs of ruminants) is relatively refractory to viral infection, with the notable exception of that overlying the tonsils, thus enteric viral infections typically begin within the mucosal epithelium of the stomach and/or intestines.
The gastrointestinal tract is protected by several different defenses, including acidity of the stomach, the layer of mucus that tenaciously covers the mucosa of the stomach and intestines, the antimicrobial activity of digestive enzymes as well as that of bile and pancreatic secretions, and innate and adaptive immune mechanisms, especially the activity of defensins and secretory antibodies such as immunoglobulin (Ig) A, the latter produced by B lymphocytes in the gastrointestinal mucosa and mucosa-associated lymphoid tissues (MALTs).
Despite these protective mechanisms, enteric infection is characteristic of certain viruses that first infect the epithelial cells lining the gastrointestinal mucosa or the specialized M cells that overlie intestinal lymphoid aggregates (Peyer’s patches).
In general, viruses that cause purely enteric infection, such as rotaviruses and enteroviruses, are acid and bile resistant. However, there are acid- and bile-labile viruses that cause important enteric infections; for example, transmissible gastroenteritis virus (a coronavirus) is protected during passage through the stomach of young pigs by the buffering action of suckled milk.
Not only do some enteric viruses resist inactivation by proteolytic enzymes in the stomach and intestine, their infectivity is actually increased by such exposure. Thus cleavage of an outer capsid protein by intestinal proteases enhances the infectivity of rotaviruses. Whereas rotaviruses and coronaviruses are major causes of viral diarrhea in animals, the great majority of enteric infections caused by enteroviruses, adenoviruses and many other viruses are typically subclinical. Some parvoviruses, morbilliviruses, amongst others, can also cause gastrointestinal infection and diarrhea, but only after reaching cells of the gastrointestinal tract in the course of generalized (systemic) infection after viremic spread.
Entry via the Skin
The skin is the largest organ of the body, and its dense outer layer of keratin provides a mechanical barrier to the entry of viruses.
The low pH and presence of fatty acids in skin provide further protection, as do various other components of innate and adaptive immunity, including the presence of migratory dendritic cells (Langerhans cells) within the epidermis itself.
Breaches in skin integrity such as insect or animal bites, cuts, punctures, or abrasions predispose to viral infection, which can either remain confined to the skin, such as the papillomaviruses, or disseminate widely.
Deeper trauma can introduce viruses into the dermis and subcutis, where there is a rich supply of blood vessels, lymphatics, and nerves that can individually serve as routes of virus dissemination. Generalized infection of the skin, such as occurs in lumpy skin disease, sheeppox, and others, is the result not of localized cutaneous infection but of systemic viral spread via viremia.
One of the most efficient ways by which viruses are introduced through the skin is via the bite of arthropods, such as mosquitoes, ticks, Culicoides spp. (hematophagous midges or “gnats”), or sandflies. Insects, especially flies, may act as simple mechanical vectors (“flying needles”); for example, equine infectious anemia virus is spread among horses, rabbit hemorrhagic disease virus and myxoma virus are spread among rabbits, and fowlpox virus is spread among chickens in this way.
However, most viruses that are spread by arthropods replicate in their vector, the defining feature of a “biological” vector. Viruses that are both transmitted by and replicate in arthropod vectors are called arboviruses. Infection can also be acquired through the bite of an animal, as in rabies, and introduction of a virus by skin penetration may be iatrogenic—that is, the result of veterinary or husbandry procedures. For example, equine infectious anemia virus has been transmitted via contaminated needles, twitches, ropes, and harnesses, and orf virus and papillomaviruses can be transmitted via ear tagging, tattooing, or virus-contaminated inanimate objects (fomites).
Entry via Other Routes
Several important pathogens (eg, several herpesviruses and papillomaviruses) are spread through the genital tract, and this is known as venereal transmission. Small tears or abrasions in the penile mucosa and the epithelial lining of the vagina may occur during sexual activity and facilitate transmission. The conjunctiva, although much less resistant to viral invasion than the skin, is constantly cleansed by the flow of secretion (tears) and mechanical wiping by the eyelids; some adenoviruses and enteroviruses, however, gain entry at this site, and a substantial number of viruses can be experimentally transmitted by this route.
Host Specificity and Tissue Tropism
The capacity of a virus to infect cells selectively in particular organs is referred to as tropism (either cell or organ tropism), which is dependent on both viral and host factors. At the cellular level, there must be an interaction between viral attachment proteins and matching cellular receptors. Although such interactions are usually studied in cultured cells, the situation is considerably more complex in vivo. Not only do some viruses require several cellular receptors/coreceptors (see Chapter 2: Virus Replication), some viruses utilize different receptors on different cells; for example, canine distemper virus uses CD150 (signaling lymphocyte activation molecule, SLAM) to infect cells of the lymphoid system, an important step in multisystemic viral spread, whereas it attaches to nectin 4 to target the epithelial cells that mediate viral shedding.
Expression of receptors can be dynamic; for example, it has been shown experimentally that animals treated with neuraminidase have substantial protection against intranasal infection with influenza virus that lasts until the neuraminidase-sensitive receptors have regenerated. Receptors for a particular virus are usually restricted to certain cell types in certain organs, and only these cells can be infected. In large part, this accounts for both the tissue and organ tropism of a given virus and the pathogenesis of the disease caused by the virus. The presence of critical receptors is not the only factor that determines whether the cell may become infected. Cells must support viral entry following receptor binding and the viral genome must be presented with factors required for transcription and genome replication. These requirements are not met by all cell types and thus represent a determinant of viral tropism. For example, paramyxoviruses may require extracellular proteases to activate their fusion protein, the fusion protein mediating viral entry following attachment. This is the case for Sendai virus, where specialized cells in the bronchioles of rats (Clara cells) secrete a protease required for productive viral infection of the lung. Similarly, papillomaviruses, retroviruses and several herpesviruses rely on the interaction between host proteins and viral genomic elements known as enhancers to support viral gene expression. Viral enhancers are gene activators that increase the efficiency of transcription of viral or cellular genes; specifically, they are short, often tandem-repeated sequences of nucleotides that may contain motifs representing DNAbinding sites for various cellular or viral site-specific DNA-binding proteins (transcription factors). Viral enhancers augment binding of DNA-dependent RNA polymerase to promoters, thereby accelerating transcription. Because many of the transcription factors affecting individual enhancer sequences in viruses are restricted to particular cells, tissues, or host species, they can determine the tropism of viruses and can act as specific virulence factors. For example, the genomic DNA of papillomavirus contains enhancers that are active only in keratinocytes and, indeed, only in the subset of these cells in which papillomavirus replication occurs.
Mechanisms of Viral Spread and Infection of Target Organs
The ability to restrict viral infection to the body surface that is the point of entry, as contrasted to multisystemic dissemination of virus infection, has profound implications on virus shedding and thus transmission of infection within a population of susceptible animals (Fig. 3.2).
From the virus’ standpoint, the challenge to local spread is the ability to infect a sufficient number of epithelial cells to support a level of shedding that assures transmission. The benefits of local spread are more limited opportunities for the immune system to disrupt the course of infection. In contrast, multisystemic spread may introduce virus to many body surfaces that can participate in shedding, and the surface area supporting replication may be much greater than can be achieved via local spread. The challenges to the virus during multisystemic spread include the numerous opportunities for the immune system to disrupt the infection cycle, the potential need to infect multiple cell types, and the need to balance cytopathic effects with the requirement for viable cells to support step-wise spread throughout the body. In pioneering experiments in 1949, Frank Fenner used ectromelia virus (the agent of mousepox) as a model system that first revealed the sequence of events leading to systemic infection and disease. Groups of mice were inoculated in the footpad of a hind limb, and at daily intervals their organs were titrated to determine the amount of virus present. Fenner showed that, during the incubation period, infection spread through the mouse body in a step-wise fashion. The virus first replicated locally in tissues of the footpad and then in the draining lymph nodes. Virus produced in these sites then gained entry into the bloodstream, causing a primary viremia, which brought the virus to its initial target organs (organ tropism), especially the spleen, lymph nodes, and the liver. Virus produced in the target organs—ie, the spleen and liver—caused a secondary viremia that disseminated virus to the skin and mucosal surfaces. Infection in the skin caused a macular and papular rash from which large amounts of virus were shed, leading to contact exposure of other mice. Infection ultimately resulted in tissue necrosis, this being the cause of death, but not until spread within the host and shedding from the host was achieved. This pattern has subsequently been demonstrated for many viruses of veterinary medical relevance, and can be illustrated by canine distemper virus infection of young immunologically naı¨ve dogs (Fig. 3.3).
FIGURE 3.3 Relationship between initiation of infection, spread, total viral load (burden), and clinical signs in a multisystemic infection, illustrated here by canine distemper virus infection of a young immunologically naı¨ve dog. Peak virus shedding occurs at the point of epithelial infection and peak viral burden in the host. Clinical signs, reflecting cumulative effects of virus replication in multiple organ systems, are not manifest until after significant virus shedding has begun. The onset of immune-mediated viral clearance correlates with the appearance of clinical signs of infection.
Following aerosol exposure, canine distemper virus replicates in lymphoid tissues associated with the respiratory tract,
resulting in primary viremia and infection of lymphoid tissues throughout the body, including the thymus and spleen. This amplifies viral burden in the host and leads to secondary viremia with infection of multiple epithelial compartments, some of which are highly efficient at viral shedding (eg, respiratory, urothelial, and conjunctival mucosa) and some of which play either a subordinate or no role in shedding and transmission (eg, integument, odontogenic epithelium, gastric mucosa). Clinical signs coincide with the peak of viral shedding, with fever signaling the onset of adaptive immune responses that drive viral clearance. Death, if it occurs, reflects the combination of immune suppression and compromised mucosal barriers that facilitate secondary microbial infections (eg, bacterial bronchopneumonia). Death may also reflect viral infection of brain, a by-product of the secondary viremia. However, these events occur only after the infection cycle is complete and shedding has occurred.
Local Spread on Epithelial Surfaces
Viruses first replicate in epithelial cells at the site of entry and produce a localized infection, often with associated virus shedding directly into the environment from these sites. The spread of infection along epithelial surfaces occurs by the sequential infection of neighboring cells, which, depending on the individual virus, may or may not precede spread into the adjacent subepithelial tissues and beyond. In the skin, papillomaviruses and poxviruses such as orf virus remain confined to the epidermis, where they induce localized proliferative lesions, whereas other poxviruses such as lumpy skin disease virus spread widely after cutaneous infection to involve other organ systems. Viruses that enter the body via the respiratory or intestinal tracts can quickly cause extensive infection of the mucosal epithelium, thus diseases associated with these infections progress rapidly after a short incubation period. In mammals, there is little or no productive invasion of subepithelial tissues of the respiratory tract after most influenza and parainfluenza virus infections, or in the intestinal tract following most rotavirus and coronavirus infections. Although these viruses apparently enter lymphatics and thus have the potential to spread, they usually do not do so, because appropriate viral receptors or other permissive cellular factors such as cleavage-activating proteases or transcription enhancers are restricted to epithelial cells, or because of other physiological constraints. Restriction of viral infection to an epithelial surface should never be equated with lack of virulence or disease severity. Although localized, injury to the intestinal mucosa caused by rotaviruses and coronaviruses can result in severe and, especially in neonates, even fatal diarrhea. Similarly, influenza virus infection can cause extensive injury in the lungs, leading to acute respiratory distress syndrome and possibly death.
Subepithelial Invasion and Lymphatic Spread
A variety of factors probably contribute to the ability of some viruses to breach the epithelial barrier and to invade the subepithelial tissues, including (1) targeted migration of virus within phagocytic leukocytes, specifically dendritic cells and macrophages, and (2) directional shedding of viruses from the infected epithelium (see Chapter 2: Virus Replication). Dendritic cells are abundant in the skin and at all mucosal surfaces, where they constitute a critical first line of immune defense, both innate and adaptive (see Chapter 4: Antiviral Immunity and Virus Vaccines). Migratory dendritic cells (such as Langerhans cells in the skin) “traffic” from epithelial surfaces to mucosa-associated lymphoid tissue (MALT), which would include lymphoid organs such as tonsils and Peyer’s patches, and the adjacent (draining) regional lymph node. Infection of these migratory dendritic cells may be responsible for the initial spread of alphaviruses, bluetongue, African horse sickness and other orbiviruses, and feline and simian human immunodeficiency viruses, amongst many others. Directional release of virus into the lumen of the respiratory or intestinal tracts facilitates local spread to the surface of contiguous epithelial cells and immediate shedding into the environment, whereas shedding from the basolateral cell surface of epithelial cells potentially facilitates invasion of subepithelial tissues and subsequent virus dissemination via lymphatics, blood vessels, or nerves. Many viruses that are widely disseminated in the body following infection at epithelial surfaces are first carried to the adjacent (regional) lymph nodes through the afferent lymphatic drainage (Fig. 3.4).
FIGURE 3.4 Subepithelial invasion and lymphatic spread of infection. Virus may be amplified in the epithelium or infect subepithelial macrophages. Cell-free virus or infected macrophages/dendritic cells pass to regional lymph nodes via afferent lymphatics, where further viral amplification may occur. Viral progeny are released into the venous circulation to cause viremia, which may be either cell-free or cell-associated.
Within the draining lymph node, virions may be inactivated and processed by macrophages and dendritic cells so that their component antigens are presented to lymphocytes to stimulate adaptive immune responses (see Chapter 4: Antiviral Immunity and Virus Vaccines). Some viruses, however, replicate efficiently in macrophages (eg, many retroviruses, orbiviruses, filoviruses, canine distemper virus and other morbilliviruses, arteriviruses such as porcine reproductive and respiratory syndrome virus, and some herpesviruses), and/or in dendritic cells and lymphocytes. From the regional lymph node, virus can spread to the bloodstream in efferent lymph, and then quickly be disseminated throughout the body, either within cells or as cell-free virions. Blood-filtering organs, including the lung, liver, and spleen, are often target organs of viruses that cause disseminated infections. Normally, there is a local inflammatory response at the site of viral invasion, the severity of which reflects the extent of tissue damage. Inflammation leads to characteristic alterations in the flow and permeability of local blood vessels, as well as leukocyte trafficking and activity. Some viruses take advantage of these events to infect cells that participate in this inflammatory response, which in turn can facilitate spread of these viruses either locally or systemically. Local inflammation may be especially important to the pathogenesis of arthropod-transmitted viruses because of the marked reaction at the site of virus inoculation induced by the bite of the arthropod vector.
Spread via the Bloodstream: Viremia
The blood is the most effective vehicle for rapid spread of virus through the body. Initial entry of virus into the blood after infection is designated primary viremia, which, although usually inapparent clinically (subclinical), leads to the seeding of distant organs. Virus replication in major target organs leads to the sustained production of much higher concentrations of virus, producing a secondary viremia (Fig. 3.5) and infection in yet other parts of the body that ultimately results in the clinical manifestations of the associated disease. In the blood, virions may circulate free in the plasma or may be contained in, or adsorbed to, leukocytes, platelets, or erythrocytes (red blood cells).
FIGURE 3.5 The role of viremia in the spread of viruses through the body, indicating sites of replication and important routes of shedding of various viruses. Subepithelial invasion and spread of infection is associated with a primary round of replication that leads to primary viremia. That viremia infects target organs that further amplify viral burden, resulting in a high-level secondary viremia. Secondary viremia may result in the infection of target organs that are conducive to viral shedding, transmission of infection via arthropod vectors, or infection of organs that are a dead end for transmission (eg, brain).
Parvoviruses, enteroviruses, togaviruses, and flaviviruses typically circulate free in the plasma. Viruses carried in leukocytes, generally lymphocytes or monocytes, are often not cleared as readily or in the same way as viruses that circulate in the plasma. Specifically, cellassociated viruses may be protected from antibodies and other plasma components, and they can be carried as “passengers” when leukocytes that harbor the virus emigrate into tissues. Individual viruses exhibit tropism to different leukocyte populations; thus monocyte-associated viremia is characteristic of canine distemper, whereas lymphocyteassociated viremia is a feature of Marek’s disease and bovine leukosis. Erythrocyte-associated viremia is characteristic of infections caused by African swine fever virus and bluetongue virus. The association of bluetongue virus with erythrocytes facilitates both prolonged viremia by delaying immune clearance, and infection of the hematophagous (blood-feeding) Culicoides midges that serve as biological vectors of the virus. A substantial number of viruses, including equine infectious anemia virus, bovine viral diarrhea virus, and bluetongue virus, associate with platelets during viremia—an interaction that might facilitate infection of endothelial cells. Neutrophils, like platelets, have a very short lifespan; neutrophils also possess powerful antimicrobial mechanisms and they are rarely infected, although they may contain phagocytosed virions.
Virions circulating in the blood are removed continuously by macrophages, thus viremia can typically be maintained only if there is a continuing introduction of virus into the blood from infected tissues or if clearance by tissue macrophages is impaired. Although circulating leukocytes can themselves constitute a site for virus replication, viremia is usually maintained by infection of the parenchymal cells of target organs such as the liver, spleen, lymph nodes, and bone marrow. In some infections, such as African horse sickness virus and equine arteritis virus infections of horses, viremia is largely maintained by the infection of endothelial cells and/or macrophages and dendritic cells. Striated and smooth muscle are an uncommon site for viral replication, not representing a target organ essential to completion of the viral infection cycle within the host, but nonetheless significant from a clinical standpoint due to the clinical signs associated with inflammation of the muscle (eg, the myositis that may accompany influenza virus infections). There is a general correlation between the magnitude of viremia generated by blood-borne viruses and their capacity to invade target tissues. Certain neurotropic viruses are virulent after intracerebral inoculation, but avirulent when given peripherally, because they do not attain viremia titers sufficient to facilitate invasion of the nervous system. The capacity to produce viremia and the capacity to invade tissues from the bloodstream are, however, two different properties of a virus. For example, some strains of Semliki Forest virus (and certain other alphaviruses) have lost the capacity to invade the CNS while retaining the capacity to generate a viremia equivalent in duration and magnitude to that produced by neuroinvasive strains. Viruses that circulate in blood, especially those that circulate free in plasma, encounter, amongst many others, two cell types that exert especially important roles in determining the subsequent pathogenesis of infection: macrophages and vascular endothelial cells.
Virus Interactions with Monocytes and Macrophages
Macrophages are bone marrow-derived mononuclear phagocytic cells that are present in all compartments of the body. Their precursors are monocytes in the blood, the largest of the leukocytes. Monocytes migrate into tissues to become part of the normal resident macrophage population found in submucosal connective tissue, spleen and bone marrow, alveoli of the lung, sinusoids of lymph nodes and liver, and parenchyma of the brain (ie, brain microglia). Monocytes also migrate into areas of inflammation to supplement the macrophage population. Macrophages are generally considered to play host protective roles in microbial infection (Fig. 3.6).
FIGURE 3.6 Types of interactions between viruses and monocytes and macrophages. Virus may exploit these cells to facilitate spread or to generate viral progeny following infection. Alternatively, macrophages may restrict virus replication and take on a host defense role which includes initiation of innate immune and pro-inflammatory responses. Innate immune responses include production of type 1 interferon (IFN) and presentation of viral antigen, both of which facilitate subsequent adaptive immunity to the virus. Pro-inflammatory responses include production of cytokines such as tumor necrosis factor (TNF). While these pro-inflammatory responses can mediate host protective responses, excesses can paradoxically contribute to manifestations of disease.
They may phagocytize and thus inactivate viruses and, together with dendritic cells, have a critical role in antigen processing and presentation to other immune cells that is central to the initiation of adaptive immune responses (see Chapter 4: Antiviral Immunity and Virus Vaccines). They also initiate innate immune responses because of their ability to detect the presence of pathogen-associated molecular patterns (“microbial signatures”) through specific receptors—eg, Toll-like receptors.
Toll-like receptor signaling is an important basis for the production of type I interferons that restrict viral virulence. In contrast to these protective roles, macrophages may contribute to the spread of virus infection and/or tissue damage. Some viruses exhibit a specific tropism for macrophages, where they replicate to high levels. Venezuelan equine encephalitis virus is one such virus, where replication of the virus in macrophages determines the level of viremia which in turn facilitates invasion of the CNS. Viral replication in macrophages may also be envisioned to reduce the contributions of these cells to innate and adaptive antiviral immune responses, thus indirectly contributing to viral burden and spread.
Productive infection of macrophages may facilitate local viral spread to neighboring parenchymal cells, as has been suggested for infectious canine hepatitis virus infection of dogs where viral antigen is detected in both hepatocytes and sinusoidal macrophages (Kupffer cells) of the liver. Virus infection of macrophages may enhance inflammatory responses that contribute to tissue injury. For example, hemorrhagic viral fevers caused by Ebola and bluetongue viruses are characterized by induction of inflammatory and vasoactive mediators such as tissue necrosis factor (TNF) by macrophages and dendritic cells, and these cytokines contribute to the pathogenesis of disease.
It should be emphasized that virus infection of macrophages may reflect interaction of viral attachment proteins with specific host cell receptors, or simply an indirect consequence of the phagocytic mechanisms employed by these cells. Although macrophages are inherently efficient phagocytes, this capacity is even further enhanced after their activation by certain microbial products and cytokines such as interferon-γ. Macrophages also have Fc receptors and C3 receptors that further augment their ability to ingest opsonized virions, specifically those virions that are coated with antibody or complement molecules. For viruses that are capable of replicating in macrophages, opsonization of virions by antibody can actually facilitate antibody-mediated enhancement of infection, which may be a major pathogenetic factor in human dengue and several retrovirus infections. Virus infection of monocytes should be considered in this discussion, having potential to profoundly influence viral spread by exploiting the tendency of these cells to migrate into tissues as part of an inflammatory response or simply to replenish the normal resident macrophage population. Monocyte infection is a form of cell-associated viremia that is important to the pathogenesis of lentivirus infections and a proposed mechanism for neuroinvasion by paramyxoviruses. In many instance, the contribution of virus interaction with macrophages is more difficult to define in terms of its protective versus detrimental role to the host. Macrophages are heterogeneous in their functional activity, which can vary markedly depending on their location and state of activation; even in a given tissue or site there are subpopulations of macrophages that differ in phagocytic activity and in susceptibility to viral infection. Differences in virusmacrophage interactions may account for differences in the virulence of closely related viruses, individual strains of the same virus, and differences in host resistance.
Virus Interactions with Vascular Endothelial Cells
The vascular endothelium with its basement membrane constitutes the bloodtissue interface and may represent a barrier for particles such as virions in locations where endothelial cells are nonfenestrated and joined together by tight junctions.
The degree of barrier function varies between tissue compartments, being greatest in the brain and eye. Parenchymal invasion by circulating virions depends on crossing such barriers, often in capillaries and venules, where blood flow is slowest and the vascular wall is thinnest.
Virions may move passively between or through endothelial cells and the basement membrane of small vessels, be carried within infected leukocytes (socalled “Trojan horse” mechanism), or infect endothelial cells and “grow” their way through this barrier, with infection of the luminal aspect of the cell and release from the basal aspect. This subject has been studied most intensively in relation to viral invasion of the CNS, but it also applies to invasion of many tissues during generalized infections. Endothelial infection may be clinically inapparent, reflecting a noncytopathic infection that facilitates viral spread.
Alternatively, infection of endothelial cells may be characterized by vascular injury that results in widespread hemorrhage and/or edema, contributing to the pathogenesis of the so-called hemorrhagic viral fevers. Virus-induced endothelial injury leads to vascular thrombosis and, if widespread, disseminated intravascular coagulation (consumptive coagulopathy). However, it is likely that inflammatory and vasoactive mediators produced by virus-infected macrophages and dendritic cells, such as tissue necrosis factor, also contribute to the pathogenesis of vascular injury in hemorrhagic viral fever (Fig. 3.6).
Spread via Nerves
Although infection of the CNS can occur after hematogenous spread, invasion via the peripheral nerves is also an important route of infection—eg, in rabies, Borna disease, and several alphaherpesvirus infections (eg, B virus encephalitis, pseudorabies, and bovine herpesvirus 5 encephalitis). Herpesviruses can travel to the CNS in axon cytoplasm and, while doing so, also sequentially infect Schwann cells of the nerve sheath. Rabies virus and Borna disease virus also travel to the CNS in axon cytoplasm, but usually do not infect the nerve sheath. Sensory, motor, and autonomic nerves may be involved in the neural spread of these viruses. As these viruses move centripetally, they must cross cellcell junctions. Rabies virus and pseudorabies virus can efficiently traverse synaptic junctions (Fig. 3.7). In addition to passing centripetally from the body surface to the sensory ganglia and from there to the brain, herpesviruses can move through axons centrifugally from ganglia to the skin or mucous membranes. This is the same phenomenon that occurs after reactivation of latent herpesvirus infections and the subsequent production of recrudescent epithelial lesions. Centrifugal spread through axons is also the mechanism by which rabies virus reaches salivary glands from the brainstem, with salivary gland infection being important to viral shedding. Viruses can also use olfactory nerve endings in the nares as sites of entry, including rhabdoviruses (eg, rabies virus and vesicular stomatitis virus), herpesviruses, and paramyxoviruses. They gain entry in the special sensory endings of the olfactory neuroepithelial cells where they cause local infection and progeny virus (or subviral entities containing the viral genome) then travel in axoplasm of olfactory nerves directly to the olfactory bulb of the brain.
Mechanisms of Virus Shedding
Shedding of infectious virions is crucial to the maintenance of infection in populations (see Chapter 6: Epidemiology and Control of Viral Diseases). For viruses that replicate only at epithelial surfaces, exit of infectious virions usually occurs from the same organ system involved in virus entry (eg, the respiratory or gastrointestinal system; Fig. 3.2).
In generalized viral infections, shedding can occur from a variety of sites (Fig. 3.5), and some viruses are shed from several sites. The amount of virus shed in an excretion or secretion is important in relation to transmission. Very low concentrations may be irrelevant unless very large volumes of infected material are involved; however, some viruses occur in such high concentrations that a minute quantity of virus-laden secretion or excretion can readily lead to transmission to the next animal host. Enteric viruses are in general more resistant to inactivation by environmental conditions than respiratory viruses; especially when suspended in water and protected from light, such viruses can persist in the environment for some time.
Viruses such as influenza and the pneumoviruses that typically cause localized infection and injury of the respiratory tract are shed in mucus and are expelled from the respiratory tract during coughing or sneezing. Viruses are also shed from the respiratory tract in several systemic infections. Enteric viruses such as rotaviruses are shed in the feces, and the more voluminous the fluid output the greater is the environmental contamination they cause. A few viruses are shed into the oral cavity from infected salivary glands (eg, rabies virus and cytomegaloviruses) or from the lungs or nasal mucosa during infection of the respiratory system. Salivary spread depends on activities such as licking, nuzzling, grooming, or biting. Virus shedding in saliva may continue during convalescence or recurrently thereafter, especially with herpesviruses. The skin is an important source of virus in diseases in which transmission is by direct contact or via small abrasions: papillomaviruses and some poxviruses and herpesviruses employ this mode of transmission. Although skin lesions are produced in several generalized diseases, the skin is not generally a source of significant viral shedding. Exceptions include vesicular diseases such as foot-and-mouth disease, vesicular stomatitis, and swine vesicular disease, where the causative viruses are produced in great quantities in vesicles within the mucosa and skin of affected animals; virus is shed from these lesions after the vesicles rupture. Localization of virus in the feather follicles is important in the shedding of Marek’s disease virus by infected chickens. Urine, like feces, tends to contaminate food sources and the environment. A number of viruses (eg, infectious canine hepatitis virus, foot-and-mouth disease viruses, and the arenaviruses) replicate in tubular epithelial cells in the kidney and are shed in urine. Canine distemper virus replicates in transitional epithelium of the renal pelvis, ureters and urinary bladder, also contributing to urinary viral shedding or “viruria.” Viruria is prolonged and common in equine rhinitis A virus infection and lifelong in arenavirus infections of reservoir rodent species; it constitutes the principal mode of contamination of the environment by these viruses. Several viruses that cause important diseases of animals are shed in the semen and are transmitted during coitus; for example, equine arteritis virus can be shed for months or years in the semen of apparently healthy carrier stallions, long after virus has been cleared from other tissues. Similarly, viruses that replicate in the mammary gland are excreted in milk, which may serve as a route of transmission—eg, caprine arthritisencephalitis virus, mouse mammary tumor virus, and some of the tick-borne flaviviruses. In salmonid fish, the fluid surrounding eggs oviposited during spawning may contain high concentrations of viruses such as infectious hemopoietic necrosis virus, which is an important mode of virus transmission in both hatchery and wild fish populations. Although not “shedding” in the usual sense of the word, blood and tissues from slaughtered animals must be considered important sources of viral contagion. Virus-laden blood is also the basis for transmission when it contaminates needles and other equipment used by veterinarians and others treating or handling sick animals. Similarly, the use of virus-contaminated fetal bovine serum can result in similar contamination of biological products.
Virus Infection Without Shedding
Many sites of virus replication might be considered “dead ends” from the perspective of natural spread. Infection of the brain may not result in shedding in the case of paramyxoviruses, although it is significant from the standpoint of clinical disease. Transmission may occur in instances where infected nervous tissues and muscle are ingested by carnivores and omnivores. Similarly, classical swine fever (hog cholera) and African swine fever have been translocated to different regions and countries through feeding garbage containing contaminated pork scraps. The prion diseases are an analogous example, where the unprecedented epizootic of bovine spongiform encephalopathy (mad cow disease) in the United Kingdom was spread widely amongst cattle by the feeding of contaminated meat and bone meal containing bovine offal that included nervous tissue. Some viruses, notably retroviruses and bovine virus diarrhea virus are also transmitted directly in the germplasm or by infection of the avian egg or developing mammalian embryo. Despite the lack of horizontal transmission, these vertically transmitted viruses accomplish the same ends as those shed into the environment—that is, transmission to new hosts and perpetuation in nature.
'Fenner's Veterinary Virology (5판)' 카테고리의 다른 글
| [Chapter 3] Pathogenesis of Viral Infections and Diseases - MECHANISMS OF VIRAL INJURY ANDDISEASE (0) | 2025.03.31 |
|---|---|
| [Chapter 2] Virus Replication - Virus Replication (0) | 2025.03.31 |
| [Chapter 2] Virus Replication - Entry and Uncoating (0) | 2025.03.08 |
| [Chapter 1] The Nature of Viruses (0) | 2025.03.08 |