203Fenner’s Veterinary Virology. DOI:© Elsevier Inc. All rights reserved.2011

10.1016/B978-0-12-375158-4.00010-9

Adenoviridae

Chapter ContentsProperties of Adenoviruses 203

Classification 203Virion Properties 205Virus Replication 206

Members of the Genus Mastadenovirus 207Canine Adenovirus 1 (Infectious Canine Hepatitis Virus) 207

Clinical Features and Epidemiology 207Pathogenesis and Pathology 208Diagnosis 208Immunity, Prevention, and Control 208

Canine Adenovirus 2 208Equine Adenoviruses 1 and 2 208Adenoviruses of Laboratory Rodents and Lagomorphs 209Primate Adenoviruses 209Mastadenoviruses of Cattle, Sheep, Goats, Camelids, and Pigs 210

Members of the Genus Aviadenovirus 210Quail Bronchitis Virus 210Hydropericardium Syndrome (Angara Disease) Virus 210Other Aviadenoviruses 210Members of the Genus Atadenovirus 210Reptilian Adenoviruses 210Cervine Adenovirus (Odocoileus Adenovirus 1) 211Egg Drop Syndrome Virus 211Other Atadenoviruses 212Members of the Genus Siadenovirus 212Turkey Adenovirus 3 (Hemorrhagic Enteritis of Turkeys,

Marble Spleen Disease of Pheasants, and Avian Adenovirus Splenomegaly Virus) 212

Other Siadenoviruses 212Other Adenoviruses 212

In 1953, Wallace Rowe and colleagues, having observed that explant cultures of human adenoids degenerated spontane-ously, isolated a new virus that they named adenovirus. The next year, Cabasso and colleagues demonstrated that the etio-logical agent of infectious canine hepatitis was an adenovirus. Subsequently, numerous adenoviruses, most appearing to be highly host specific, were isolated from humans and many other mammals and birds, usually from the upper respiratory tract, but sometimes from feces. Indeed, it is likely that all ver-tebrate species, from fish to mammals, have their own unique adenovirus or adenoviruses with which they have co-evolved. Most of these viruses produce subclinical infections in their respective hosts, with occasional upper respiratory disease, but canine and avian adenoviruses are especially associated with clinically important disease syndromes.

Since their discovery, adenoviruses have been at the core of significant basic discoveries concerning virus structure, eukaryotic gene expression and organization, RNA splicing, and apoptosis. Adenoviruses are frequently used as experi-mental vectors for gene therapy and cancer therapy, and have been used as vectors for recombinant vaccines. They also received a brief flurry of interest shortly after their dis-covery, because of their oncogenic behavior in experimen-tally infected laboratory rodents. Specifically, some of the

adenoviruses of humans, cattle, and chickens cause tumors when inoculated into newborn laboratory animals and have been used in experimental oncogenesis studies; however, none has been proven to cause tumors in their respective natural hosts.

PrOPerties Of AdenOviruses

Classification

The family Adenoviridae currently comprises four serologi-cally distinct genera: (1) the genus Mastadenovirus, com-prising viruses that infect only mammalian species; (2) the genus Aviadenovirus, comprising viruses that infect only birds; (3) the genus Atadenovirus that includes viruses that infect a broad host range, including snakes, lizards, ducks, geese, chickens, possums, and ruminants; (4) the genus Siadenovirus, which includes frog adenovirus 1 and turkey adenovirus 3, plus several recently described viruses of rap-tors, budgerigars, and tortoises. A fifth genus that includes adenoviruses of fish such as white sturgeon adenovirus is proposed. Although all adenoviruses have a similar morphol-ogy (Figure 10.1), the genomic organization differs between

Chapter 10

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viruses in the various genera (Figure 10.2). Mastadenoviruses contain the unique proteins V and IX; protein V is involved in transport of viral DNA to the cell nucleus and protein IX is a transcriptional activator. Genes encoding proteins V and IX are absent in aviadenoviruses and their genomes are 20–45% larger than those of mastadenoviruses. Atadenoviruses encode a unique structural protein, p32K, and apparently lack the immunomodulatory proteins that occur in the E3 region of mastadenoviruses. The genomic structure of siadenovi-ruses is also unique in that genes encoding proteins V and IX are absent as well as the genes encoding early regions E1, E3, and E4 of mastadenoviruses.

Adenoviruses are designated by their host species and a serial number (e.g., canine adenovirus 1), as listed in Table 10.1. Genomic organization and relatedness, growth characteristics in cell culture, and host range have all been used for the pre-cise categorization of virus strains and, in general, results have accorded well with previous categorizations based on serologi-cal cross-reactions. After the general structuring of the fam-ily had been re-done on the basis of molecular characteristics

of the viruses, the basis for the immunologic relationships among the viruses became clear. Specifically, antigenic deter-minants associated with the inner part of hexons—that is, the structural units making up the bulk of the capsid—contain the epitopes that were first used antigenically to define the two original genera. Hexons are involved in neutralization, and fib-ers in both neutralization and hemagglutination (Figure 10.1). Genus-specific antigen is located on the basal surface of the hexon, whereas serotype-specific antigens are located mainly on the tower region of the hexon. Serotypes are differentiated on the basis of neutralization assays; they are defined as those that include adenoviruses that exhibit no cross-reaction with other adenoviruses or show a homologous/heterologous titer ratio, in both directions, of greater than 16. The penton fibers contain other type-specific epitopes, which are also important in neutralization assays. Unexpectedly, although the distal (fiber) knobs on the penton contain the cell-binding ligands that are responsible for virus attachment to specific cellular recep-tors, antibody to these knobs or to the penton fibers is only weakly neutralizing. Thus the previous serologic structuring of

TABLE 10.1 Diseases of Domestic Animals Associated with Adenoviruses

Animal Species

Number of Serotypes

Disease

Dogs 2 Infectious canine hepatitis (canine adenovirus 1)Infectious canine tracheobronchitis (canine adenovirus 2)

Horses 2 Usually asymptomatic or mild upper respiratory disease. Bronchopneumonia and generalized disease in Arabian foals with primary severe combined immunodeficiency disease

Cattle 10 Usually asymptomatic or mild upper respiratory disease; occasionally severe respiratory or enteric disease in calves

Swine 4 Usually asymptomatic or mild upper respiratory disease

Sheep 7 Usually asymptomatic or mild upper respiratory disease; occasionally severe respiratory or enteric disease in lambs

Goats 2 Usually asymptomatic or mild upper respiratory disease

Deer 1 Pulmonary edema, hemorrhage, vasculitis

Rabbits 1 Diarrhea

Chickens 14 12 serotypes of aviadenovirus: fowl adenoviruses 1–11 and 8a and 8b (hydropericardium syndrome, inclusion body hepatitis)1 serotype of atadenovirus: egg drop syndrome1 serotype of siadenovirus: adenovirus-associated splenomegaly

Turkeys and pheasants

3 Siadenovirus: hemorrhagic enteritis (turkey); marble spleen disease (pheasant); egg drop syndrome (both)Aviadenovirus: turkey adenoviruses 1 and 2 (depressed egg production)

Quail 1 Aviadenovirus: bronchitis

Ducks 2 Atadenovirus: duck adenovirus 1 (asymptomatic or egg drop)Aviadenovirus: duck adenovirus 2 (rarely, hepatitis)

Geese 3 Aviadenovirus: isolated from liver, intestines

Chapter | 10 Adenoviridae 205

the family was based more on the relative dominance of certain epitopes in particular serological tests than on their location in the virion.

virion Properties

Adenovirus virions are non-enveloped, precisely hexagonal in outline, with icosahedral symmetry, 70–90 nm in diam-eter (Figure 10.1; Table 10.2). Virions are composed of 252 capsomers: 240 hexons that occupy the faces and edges of the 20 equilateral triangular facets of the icosahedron and 12 pentons (vertex capsomers) that occupy the vertices. The hexons consist of two distinct parts: a pseudohexagonal base with a hollow center, and a triangular top that includes three distinct “towers.” From each penton projects a penton fiber 9–77.5 nm in length, with a terminal knob. Avian ade-noviruses have two fiber proteins per vertex.

The genome of adenoviruses consists of a single linear molecule of double-stranded DNA, 26–45 kbp in size, with inverted terminal repeats. The viral genome encodes approxi-mately 40 proteins that are transcribed after complex RNA splicing. About one-third of the proteins are structural pro-teins, including a virus-encoded cysteine protease that is nec-essary for processing of some precursor proteins. Structural proteins include those that make up the hexons, pentons, and penton fibers, and others associated with the virion core.

Many adenoviruses agglutinate red blood cells, with hemagglutination occurring when the tips of penton fibers bind to cellular receptors and form bridges between cells. The optimal conditions and species of red blood cells for demonstrating this phenomenon with each adenovirus have been determined, as the hemagglutination-inhibition assay (see Chapter 5) has been a major serologic diagnostic method for many years.

II

III

IVIIlaVVIVIIVIIIIXX

DNA

TP

fiGure 10.1 (Left) Cryo-electron reconstruction of a particle of an isolate of human adenovirus 2 (Stewart et al. (1991). Cell, 67:145–154). (Center). Stylized section of a mastadenovirus particle showing capsid (II, III, IIIa, IV, VI, VIII, IX) and core (V, VII, X and TP (terminal protein)) proteins. As the structure of the nucleoprotein core has not been established, the polypeptides associated with the DNA are shown in hypothetical loca-tions. (Adapted from Stewart, P.L. and Burnett, R.M. (1993). Jpn. J. Appl. Phys., 32, 1342–1347). (Right) Negative contrast electron micrograph of a particle of an isolate of human adenovirus 2 (Valentine, R.C. and Pereira, H.G. (1965). J. Mol. Biol., 13, 13–20). [From Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses (C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, L. A. Ball, eds.), p. 213. Copyright © Elsevier (2005), with permission.]

TABLE 10.2 Properties of Adenoviruses

Four genera: Mastadenovirus, Aviadenovirus, Atadenovirus, and Siadenovirus

Virions are non-enveloped, hexagonal in outline, with icosahedral symmetry, 70–90 nm in diameter, with one (genus Mastadenovirus) or two (genus Aviadenovirus) fibers (glycoprotein) projecting from each vertex of the capsid

The genome consists of a single linear molecule of double-stranded DNA, 26–45 kbp in size, with inverted terminal repeats

Replication takes place in the nucleus by a complex program of early and late transcription (before and after DNA replication); virions are released by cell lysis

Intranuclear inclusion bodies are formed, containing large numbers of virions, often in paracrystalline arrays

Viruses agglutinate red blood cells

Some viruses are oncogenic in laboratory animals

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Adenoviruses are relatively stable in the environment, but are inactivated easily by common disinfectants. Most of the viruses have narrow host ranges; however, canine adenovirus 1, the cause of infections canine hepatitis, has also caused epizootics in foxes, bears, wolves, coyotes, and skunks. Many adenoviruses cause acute respiratory or gas-troenteric disease of varying severity.

virus replication

Adenoviruses replicate in the nucleus, and their replication is facilitated by extensive modulation of the host immune response. Viruses bind to host-cell receptors via their penton fiber knobs and subsequent internalization is mediated by the interaction between the penton base and cellular integrins. The outer capsid is then removed and the core comprising the viral genome with its associated histones enters the nucleus where messenger RNA (mRNA) transcription, viral DNA replica-tion, and assembly of virions occur (see also Figure 2.10).

In the nucleus, the genome is transcribed by cellular RNA polymerase II according to a complex program involving both DNA strands (Figure 10.2). There are five early (E) transcriptional units (E1A, E1B, E2, E3, and E4), two intermediate units (IX and IVa2), and one late (L) unit from which five families of late mRNAs (L1 to L5) are tran-scribed. Each early region is under the control of a separate promoter, whereas the late region uses a single promoter called the major late promoter. The E1A region of the viral genome encodes proteins that are essential for three main outcomes of early adenovirus transcription: (1) induction of cell-cycle progression (DNA synthesis) to provide an optimal environment for virus replication; (2) protection of infected cells from host antiviral immune defenses, includ-ing cytokine-induced apoptosis; (3) synthesis of viral pro-teins necessary for viral DNA replication.

E1A and E1B gene products are also responsible for cell transformation and hence for the oncogenicity (experimental) of some adenoviruses. Both proteins inactivate the cellular

fiGure 10.2 Schematic illustration of the different genome organizations found in members of the four genera of adenoviruses. Black arrows depict genes conserved in every genus, gray arrows show genes present in more than one genus, colored arrows shows genus-specific genes. HAdV-2, human adenovirus 2; FAdV-1, fowl adenovirus 1; OAdV-7, ovine adenovirus 7; TAdV-3, turkey adenovirus 3. [From Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses (C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, L. A. Ball, eds.), p. 214. Copyright © Elsevier (2005), with permission.]

Chapter | 10 Adenoviridae 207

tumor suppresser gene, p53, and thus deregulate cell-cycle progression. Inactivation is mediated by ubiquitination of p53 and other proteins through virus-assembled E3 ligases, leading to proteasome-mediated degradation. The E3 region is not essential for adenovirus replication in cell cultures, and can be deleted or replaced without disrupting virus replica-tion in vitro. It is therefore one of the insertion sites for for-eign DNA when constructing adenovirus vectors. E3 proteins are known to interact with host immune defense mechanisms, thus modulating the host response to adenovirus infection. Inhibition of class I major histocompatibility antigen trans-port by E3/19K inhibits recognition of infected cells by cyto-toxic T lymphocytes and natural killer cells. Tumor necrosis factor-induced apoptosis is inhibited by adenoviral E3/14.7K through the blocking of tumor necrosis factor receptor 1 internalization, which prevents establishment of the death-inducing signaling complex. E3/14.7K has also been shown to modulate antiviral inflammatory responses by inhibiting nuclear factor B (NFB) transcriptional activity.

Viral DNA replication, using the 5-linked 55K protein as primer, proceeds from both ends by a strand-displacement mechanism. The repeat sequences form panhandle-like structures of single-stranded DNA that serve as origins of replication. After DNA replication, late mRNAs are tran-scribed; these are translated into structural proteins, which are made in considerable excess. All adenovirus late-coding regions are transcribed from a common promoter, the major late promoter. The primary transcript is about 29 kb; at least 18 distinct mRNAs are produced by alternative splicing of the late primary transcript. Shutdown of host-cell macromo-lecular synthesis occurs progressively during the second half of the replication cycle. Virions are assembled in the nucleus, where they form crystalline arrays (as shown in (Figure 2.2) Chapter 2). Many adenoviruses cause severe condensation

and margination of the host-cell chromatin, making nuclei appear abnormal; this is the basis for the inclusion bodies seen characteristically in adenovirus-infected cells (Figure 10.3). Virions are released by cell lysis.

MEMBErs of ThE gEnus Mastadenovirus

CAnine AdenOvirus 1 (infeCtiOus CAnine HePAtitis virus)

Infectious canine hepatitis, a systemic disease caused by canine adenovirus 1, is also an important pathogen of foxes, wolves, coyotes, skunks, and bears. In fact, the virus was first recognized as the cause of fox encephalitis. In dogs, as well as causing acute hepatitis, the virus may cause respiratory or ocular disease. In contrast, canine ade-novirus 2 infection is localized to the respiratory tract (as described in the subsequent section).

Clinical features and epidemiology

Disease induced by canine adenovirus 1 is well controlled by vaccination in many countries and, therefore, most infections are asymptomatic or manifest as undifferentiated respiratory disease. In some cases, especially in the immunologically naïve host, the infection proceeds from the initial respiratory site to cause systemic disease. The systemic disease may be divided into three overlapping syndromes, which are usually seen in younger animals: (1) peracute disease in which the pup is found dead either without apparent preceding illness or after an illness lasting only 3 or 4 hours; (2) acute disease, which may be fatal, marked by fever, depression, loss of appetite, vomiting, bloody diarrhea, petechial hemorrhages of the gums, pale mucous membranes, and icterus (jaundice); (3) mild disease, which may actually be a vaccine-modified disease—that is, the result of partial immunity.

The incubation period of the acute disease is 4–9 days. Clinical signs include fever, apathy, anorexia, thirst, conjunc-tivitis, serous discharge from the eyes and nose, and occasion-ally abdominal pain and petechiae of the oral mucosa. There is tachycardia, leukopenia, prolonged clotting time, and dissemi-nated intravascular coagulation. In some cases there is hemor-rhage (e.g., bleeding around deciduous teeth and spontaneous hematomas). Although central nervous system involvement is not common, dogs affected severely may convulse. Seven to 10 days after acute signs disappear, about 25% of affected dogs develop a characteristic and diagnostically useful bilat-eral corneal opacity, which usually disappears spontaneously.

In foxes, canine adenovirus 1 causes primarily cen-tral nervous system disease; infected animals may exhibit intermittent convulsions during the course of their illness and, terminally, may suffer paralysis of one or more limbs.

Infection of the kidney is associated with viruria, which is a major mode of transmission, along with feces and

fiGure 10.3 Avian adenovirus 1 infection in the spleen of a chick. The nucleus at the left contains dispersed virions and early margination of chromatin, whereas the nucleus at the right contains many virions and extremely condensed chromatin. Thin-section electron microscopy; mag-nification 16,000. (Courtesy of N. Cheville.)

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saliva. Recovered dogs may shed virus in their urine for up to 6 months.

Pathogenesis and Pathology

The virus enters through nasopharyngeal, oral, and conjunc-tival routes; initial infection occurs in tonsillar crypts, spread-ing to regional lymph nodes and to the blood via the thoracic duct. Viremia results in dissemination to saliva, urine, feces, and infection of endothelial and parenchymal cells in many tissues, leading to hemorrhages and necrosis, especially in the liver, kidneys, spleen, and lungs. Canine adenovirus 1 is also one of the several causes of acute respiratory disease, although it is probably less important than canine adenovirus 2. The syndrome that gave the disease its name, infectious canine hepatitis, involves the extensive destruction of hepa-tocytes, resulting in peracute death. Invariably in such cases, histologic examination reveals characteristic inclusion bodies in hepatocytes.

In the convalescent stages of natural infection and 8–12 days after vaccination with canine adenovirus 1 attenuated-virus vaccine, corneal edema (“blue eye”) is occasionally observed. Although clinically dramatic and alarming, espe-cially after vaccination, the edema usually resolves after a few days, without consequence. The edema is caused by virus–antibody complexes (type III immune complex hypersensitiv-ity), deposited in the small blood vessels of the ciliary body, interfering with normal fluid exchange within the cornea.

Pathologic findings depend on the clinical course of infection. A rapid clinical course results in edema and hem-orrhage of superficial lymph nodes, with multifocal to dif-fuse petechial and ecchymotic hemorrhages on serosal surfaces. The liver and spleen are enlarged, with mottling of the splenic parenchyma, and accumulation of fibrin on the serosal surfaces of the abdominal viscera. The wall of the gallbladder is characteristically thickened and edematous. Gross lesions in other organs may include cortical renal hemorrhages and multiple areas of pulmonary consolidation. Ocular lesions may include diffuse corneal edema and opac-ity. Histologic hepatic findings in acutely infected puppies include multifocal hepatocellular necrosis, and sometimes centrilobular hepatic necrosis as a consequence of dissemi-nated intravascular coagulation. Intranuclear inclusions may be present within Kupffer’s cells and hepatocytes. Viral inclusions also occur in endothelial cells within the kidney of affected dogs. There is typically widespread hemorrhage and necrosis associated with intravascular thrombosis in dogs that develop disseminated intravascular coagulation.

diagnosis

Diagnosis of canine adenovirus infections is achieved by either virus isolation or serology using an enzyme immu-noassay, hemagglutination-inhibition, or neutralization assay. Viral DNA can be detected directly by polymerase

chain reaction (PCR) assay. Virus isolation is performed in any of several cell lines of canine origin (e.g., Madin–Darby canine kidney cells). Cytopathology occurs in most cases in 24–48 hours and, in addition to the characteristic intranuclear inclusions, canine adenovirus 1 can be identified by immu-nohistochemistry and/or immunofluorescence. Virus persists in renal tubular epithelium cells and therefore can be isolated from urine for months after resolution of clinical signs.

immunity, Prevention, and Control

Both inactivated and attenuated canine adenovirus 1 vaccines have been widely used for many years. The antigenic rela-tionship between canine adenoviruses 1 and 2 is sufficiently close for canine adenovirus 2 vaccine to be cross-protective; it has the advantage that it does not cause corneal edema. Annual revaccination is recommended by many manufactur-ers. Maternal antibody interferes with active immunization until puppies are 9–12 weeks of age. The development of neutralizing antibodies directly correlates with immune pro-tection, and dogs with high neutralization titers are protected against clinical disease.

One of the most remarkable phenomena in veterinary practice has been the virtual disappearance of infectious canine hepatitis from regions where vaccination had been performed for many years. This may in part be a result of the shedding of vaccine virus by vaccinated dogs, thereby “seeding” the environment with attenuated virus, immuniz-ing other dogs secondarily, and building up a high level of herd immunity.

CAnine AdenOvirus 2

Canine adenovirus 2 causes a localized respiratory disease in dogs and is a potential cause of the kennel cough syndrome (acute respiratory disease of canines). Respiratory disease in affected dogs is characterized principally by bronchitis and bronchiolitis. An essential difference between canine adeno-viruses 1 and 2 is that, whereas canine adenovirus 1 causes systemic disease, canine adenovirus 2 infection results only in restricted respiratory disease. The molecular basis of this difference remains uncertain, but this property is exploited for vaccination of dogs: specifically, although the use of live-attenuated canine adenovirus 1 vaccines sometimes results in blue eye because of the ability of the vaccine virus to rep-licate systemically, canine adenovirus 2 vaccines do not rep-licate systemically. Canine adenovirus 2 vaccines, however, provide complete homologous and cross protection against disease induced by canine adenovirus 1.

equine AdenOviruses 1 And 2

Two equine adenoviruses, equine adenoviruses 1 and 2, have been identified. Equine adenovirus 1 has been iso-lated worldwide from upper respiratory secretions of foals

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and horses with and without disease. Equine adenovirus 2 has been isolated from lymph nodes and feces of foals with upper respiratory disease and diarrhea. Most equine adeno-virus infections are asymptomatic or present as mild upper or lower respiratory tract disease. The latter are marked by fever, nasal discharge, and cough. Secondary bacterial infections, which produce a mucopurulent nasal discharge and exacerbate the cough, are not uncommon.

Arabian foals that have genetically based (defective V(D)J recombination) primary severe combined immuno-deficiency disease, an autosomal inherited defect in which there is a total absence of both T and B cells, are incapable of mounting an adaptive immune response to equine adeno- viruses. As maternal antibody wanes, these foals become susceptible to adenovirus infection. Infection is progres-sive, and these foals invariably die within 3 months of age. Much research has been done on adenovirus infections in Arabian foals with primary severe combined immu-nodeficiency disease. Among all the potentially important opportunistic pathogens that may take advantage of the immune incompetence of these foals, the dominant role of equine adenovirus 1 in the overall pathogenesis of this syndrome is intriguing. In addition to causing bronchiolitis and pneumonia, the virus destroys cells in a wide range of other tissues in these foals, particularly the pancreas and salivary glands, but also renal, bladder, and gastrointestinal epithelium.

A diagnosis of adenovirus infection can, in most cases, be made by virus isolation, serology, or PCR detection and analysis of viral nucleic acid. Adenovirus antigen detection using enzyme immunoassay and virus-specific monoclonal antibodies may also be used. Virus isolation (from nasal swabs of suspect cases or tissues of foals with primary severe combined immunodeficiency disease) is performed in any of several cell lines of equine origin. Cytopathology typical of adenovirus infections (rounding and grape-like clustering of infected cells) occurs in most cases in 24–48 hours. Serologic diagnosis is usually made by hemagglutination-inhibition or neutralization tests. A vari-ety of nucleic acid detection methods have been described, including DNA restriction endonuclease mapping (fin-gerprinting), Southern blot, dot-blot, in-situ hybridization and, most recently, PCR assay. Of these, the PCR assay is becoming the most widely used.

Like most other adenoviruses, equine viruses are proba-bly transmitted by oral and nasopharyngeal routes. Nothing is done to prevent or control infections, given their self-limiting nature.

AdenOviruses Of LAbOrAtOry rOdents And LAGOMOrPHs

Laboratory and wild mice (Mus musculus) are suscep-tible to two serologically distinct adenoviruses, known

as mouse adenoviruses 1 and 2, and previously referred to as FL virus and K87 virus, respectively. Murine ade-novirus 1 was isolated from the spleens of mice infected with Friend leukemia virus (thus the “FL” designation), and induces a multisystemic infection when inoculated into neonatal or immunodeficient mice. Naturally occur-ring disease appears to be non-existent, and the virus is very rare, if not extinct, in contemporary mouse colonies. A serologically related, but distinct adenovirus, murine adenovirus 2, is relatively more common, and may be associated with infant mouse runting and low mortality. Murine adenovirus 2 is enterotropic, producing adenoviral inclusions in enterocytes lining the villi of the small intes-tine. These inclusions are most apparent in infant mice, but may also be encountered in smaller numbers in adult mice. The serologic relatedness of these viruses involves a one-way cross-reactivity, with antibody against both murine adenoviruses 1 and 2 reacting with murine adenovirus 2 antigen, whereas antibody to murine adenovirus 2 does not react against murine adenovirus 1 antigen. Laboratory rats may also have intestinal adenoviral inclusions, and sero-convert to murine adenovirus 2, but the rat virus appears to be distinct from that of the mouse, as it is infectious only to rats. Syrian hamsters are also susceptible to an unchar-acterized intestinal adenovirus, which is probably of rat origin.

Guinea pigs are susceptible to a respiratory adenovirus that causes pulmonary disease and inclusions in respiratory epithelium of young guinea pigs. Affected animals may be severely dyspneic, with high mortality, but morbidity within a population of guinea pigs is low. Disease cannot be reproduced by experimental inoculation of guinea pigs, so other susceptibility factors are suspected in natural dis-ease. Enteritis with profuse diarrhea in young Oryctolagus rabbit kits has been described in Europe. Virus was isolated from several organs. There is evidence of seroconversion to adenovirus in North American rabbits, but no disease has been reported.

PriMAte AdenOviruses

There are numerous isolates of human adenovirus, which are now classified into six major species (human adenovi-ruses A–F). Adenoviruses of non-human primates are less well characterized, but are represented by at least 27 dis-tinct serotypes that have been isolated from a wide variety of monkeys and apes, including macaques, vervet mon-keys, baboons, gorillas, squirrel monkeys, tamarins, and chimpanzees. Most infections are subclinical, but respira-tory disease, conjunctivitis, segmental ileitis, pancreatitis, and hepatitis, all with characteristic adenoviral inclusions, have been reported in various species. The non-human pri-mate adenoviruses are genetically disparate from human adenoviruses, but serologically related.

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MAstAdenOviruses Of CAttLe, sHeeP, GOAts, CAMeLids, And PiGs

The importance of mastadenoviruses in agriculturally important domestic animals is conjectural. Several serotypes of bovine adenoviruses have been isolated from calves with pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, and weak calf syndrome. In sheep, adenoviruses are most often isolated from lambs and can be associated with respiratory and enteric infections/disease. Porcine adenoviruses have been associated with respiratory and/or enteric infection/dis-ease or encephalitis; however, it is currently believed that porcine adenoviruses rarely cause severe disease. Protracted excretion of adenoviruses in feces has been described after experimental infections, including infections with those viruses that cause respiratory disease.

The classification of bovine adenoviruses is especially complicated because of the lack of genus-specific antigens. Therefore, some bovine adenoviruses (Subgroup I: bovine ade-noviruses 1–3, 9, and 10) belong to the genus Mastadenovirus, whereas others (Subgroup II; bovine adenoviruses 4–8) are now classified in the genus Atadenovirus, which also includes ovine adenovirus 7 and caprine adenovirus 1. Ovine adeno-viruses 1–5 are included in the genus Mastadenovirus, as is, tentatively, ovine adenovirus 6. Porcine adenoviruses 1–3 and, tentatively, caprine adenovirus 2 are also included in the genus Mastadenovirus. The sequence of adenoviruses isolated from camelids with enteric disease, pneumonia, and hepatitis place them in the genus Mastadenovirus, distinct from bovine and ovine isolates. These represent either camelid adenoviruses or spill-over viruses from some unidentified contact species. Clearly, additional epidemiological and experimental studies are needed to better define the virulence and pathogenesis of the adenoviral infections of livestock.

MEMBErs of ThE gEnus aviadenovirusAviadenoviruses infect only birds, and are serologically dis-tinct from the adenoviruses in other genera. They are associ-ated with a variety of important disease syndromes in birds. The role of most aviadenoviruses as pathogens is not well defined, with the notable exception of quail bronchitis and hydropericardium syndrome viruses. Aviadenoviruses were previously classified as Subgroup I avian adenoviruses, and include fowl adenoviruses 1–11, duck adenovirus 2, pigeon adenovirus, and turkey adenoviruses 1 and 2.

quAiL brOnCHitis virus

Quail bronchitis is an important disease of wild and captive-bred bobwhite quail worldwide; in young birds it is seen as respiratory distress, open-mouth breathing, nasal discharge, coughing, sneezing, rales, lacrimation, and conjunctivitis.

In older birds there is also diarrhea. Mortality may be 100% in young birds, but falls to less than 25% in birds aged more than 4 weeks when infected. The disease is marked by tra-cheitis, air sacculitis, and gaseous, mucoid enteritis. The etiologic agent is avian adenovirus 1, which can be isolated readily from the respiratory tract of acutely affected birds and from the intestinal tract of mildly affected birds. The virus is highly contagious and spreads rapidly through flocks. Control is based on strict isolation, quarantine of introduced birds, and regular decontamination of premises and equipment. In some instances, recovered birds are retained as breeders, as there is no long-term shedding and immunity is long lasting.

HydrOPeriCArdiuM syndrOMe (AnGArA diseAse) virus

Infectious hydropericardium syndrome first appeared in 1987 in broiler fowl in Pakistan, and has spread throughout the Middle East and parts of central and eastern Asia. A milder variant of the disease has been reported in Central and South America. The disease is typically associated with infection by fowl adenovirus type 4, but the most severe manifesta-tions of the disease require co-infection with an immunosup-pressive agent or exposure to immunosuppressive aflatoxins. The disease causes 20–80% mortality, usually beginning in birds aged 3 weeks of age, and peaking at 4–5 weeks in meat chickens. A milder disease can occur in older chickens such as breeders and layers. Affected birds exhibit pericardial effusion, pulmonary edema, and hepatomegaly, and have enlarged kidneys. A vaccine is available in some countries.

OtHer AviAdenOviruses

Various disease syndromes have been associated with aviad-enovirus infections, but experimental studies are lacking and, in some instances, experimental infections with these viruses have failed to reproduce the associated disease without sec-ondary infections. Such syndromes include inclusion-body hepatitis, gizzard erosions, reduced egg production or growth rate, tenosynovitis, and respiratory disease in chickens. Similar infections by aviadenoviruses have been reported in turkeys, geese, ducks, pigeons, and ostriches. Pancreatitis has been associated with aviadenovirus infection in guinea fowl.

MEMBErs of ThE gEnus atadenovirusMembers of the genus Atadenovirus have a broad host range that includes reptiles, birds, and mammals.

rePtiLiAn AdenOviruses

Adenoviruses included in the genus Atadenovirus genus have been described in many reptile species, including

Chapter | 10 Adenoviridae 211

several different species of snake, lizards (including emer-ald monitor, Mexican beaded lizard, bearded dragon, and Gila monster), chameleons, and crocodiles. Lesions in rep-tiles include hepatitis, esophagitis, enteritis, splenitis, and encephalopathy, often with characteristic adenoviral inclu-sions in affected tissues.

Cervine AdenOvirus (OdOCOiLeus AdenOvirus 1)

In 1993, a novel adenovirus was determined to be the cause of an epizootic of severe systemic disease in mule deer (Odocoileus hemionus) in California. The causative virus, cervine adenovirus (odocoileus adenovirus 1) has tentatively been classified in the genus Atadenovirus. The disease caused by this virus also occurs amongst deer in Oregon, and in other regions of North America. Odocoileus adenovirus 1 has been isolated from naturally infected wild and/or captive white-tailed deer, mule deer, black-tailed deer, and moose, often in association with a fatal hemorrhagic disease syndrome. The disease is marked by pulmonary edema and erosions, ulcera-tions, hemorrhage, and abscesses of the intestinal tract (Figure 10.4A). Histologically, there is widespread vasculitis with endothelial intranuclear inclusions (Figure 10.4B). Laboratory diagnosis is based on the detection of viral antigen in tissues by immunofluorescence and by the detection of virions by electron microscopy or virus-specific PCR assay.

eGG drOP syndrOMe virus

Egg drop syndrome, first reported in 1976, is character-ized by the production of soft-shelled and shell-less eggs by apparently healthy chickens. The disease has been rec-ognized in fowl, and in both wild and domestic ducks and

geese worldwide, although the disease is not present in the United States. The virus originated in ducks and spread to chickens through a contaminated vaccine. Chickens are the major species affected by the disease. The virus grows to high titers in embryonating eggs of ducks or geese, or cell cultures derived from ducks, geese, or chickens—especially well in duck kidney, duck embryo liver, and duck embryo fibroblasts.

In chicken flocks without prior experience of these viruses, the first clinical signs of infection are loss of color in pigmented eggs and soft-shelled, thin-shelled, and shell-less eggs. Thin-shelled eggs may have a rough or even sandpaper-like surface. Because birds tend to eat the shell-less eggs, they may be missed, but egg production numbers decrease by a maximum of 40%. In flocks in which there is antibody, the disease is seen as a failure to achieve produc-tion targets. There is also an enzootic form of the disease, similar but more difficult to detect. Major lesions in infected birds are seen in the pouch shell gland and oviduct, where epithelial cells become necrotic and contain intranuclear inclusion bodies. There is associated inflammatory infil-tration. These findings are virtually pathognomonic, but diagnosis may be confirmed by virus isolation or serology. Hemagglutination-inhibition or neutralization assays are specific for this virus and do not cross-react with antibodies from aviadenovirus infections.

The main route of transmission is through contaminated eggs. Droppings also contain virus, and contaminated fomites such as crates or trucks can spread virus. The virus is also transmitted by needles used for vaccinations. At one time these viruses were spread by the contamination of Marek’s disease vaccine, which was produced in duck embryo fibroblasts. Breeding flocks were infected and the viruses were spread widely through fertile eggs. Because

(A) (B)fiGure 10.4 Cervine adenovirus infection. (A) Severe pulmonary edema in experimentally infected black-tailed deer (Odocoileus hemionus). (B) Intranuclear inclusions in endothelial cells (arrow) lining an affected arteri-ole. (Courtesy of L. Woods, CA Animal Health and Food Safety Laboratory.)

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infection usually remained latent until birds reached sexual maturity and because the viruses are transmitted vertically in eggs, the detection of this source of contagion was very difficult. Sporadic outbreaks have also been traced to con-tact of chickens with domestic ducks or geese, and to water contaminated with wildfowl droppings.

This disease has been eradicated from primary breeder flocks in most countries. Its entry into layer flocks is further managed by: (1) preventing contact with other birds, espe-cially waterfowl; (2) disinfecting all equipment regularly; (3) chlorination of water. Inactivated vaccines are available for use in chickens before they begin laying eggs, but they only reduce, rather than eliminate, virus transmission.

OtHer AtAdenOviruses

As described previously, the classification of ruminant adenoviruses is somewhat confusing, with some members being classified in the genus Atadenovirus (bovine adeno- viruses 4–8, ovine adenovirus 7, goat adenovirus 1), whereas the majority are included in the genus Mastadenovirus. The pathogenic significance of many of these viruses awaits definitive characterization.

MEMBErs of ThE gEnus siadenovirusThe genus Siadenovirus includes viruses that infect amphibians, birds, and reptiles.

turkey AdenOvirus 3 (HeMOrrHAGiC enteritis Of turkeys, MArbLe sPLeen diseAse Of PHeAsAnts, And AviAn AdenOvirus sPLenOMeGALy virus)

Several important disease syndromes of different bird spe-cies are caused by siadenovirus (Subgroup II avian adeno-viruses) infections. Hemorrhagic enteritis is a common acute infection of turkeys older than 4 weeks; it is characterized by splenomegaly and intestinal hemorrhage. Clinically, the disease is characterized by acute onset, depression, bloody droppings, and death. Infection causes both humoral- and cell-mediated immunosuppression, so opportunistic bacte-rial infections are often an intercurrent problem. Flock mor-tality may reach 60%, although the usual mortality is 1–3%.

A serologically indistinguishable virus causes marble spleen disease of pheasants and avian adenovirus splenomegaly in broiler chickens.

The lesions are pathognomonic: there is prominent retic-uloendothelial hyperplasia and intranuclear inclusion bodies in the spleen, distended bloody intestines, and pseudomem-branous (fibrinonecrotic) inflammation in the duodenum. Diagnosis of infection may be confirmed by serology using an immunoassay or agar gel immunodiffusion, or by virus isolation with identification of the isolates by immunohisto-chemistry, immunofluorescence or PCR assay.

The virus is transmitted readily by contact and fomites, and is very stable in contaminated droppings, litter, etc. Control of the disease in turkeys or pheasants is based on vaccination, using an attenuated virus produced either in turkey spleen cells or in turkey B lymphoblastoid cells. Vaccine is administered via drinking water. Because mater-nal antibody interferes with vaccination, the optimum age for vaccination (usually 4–5 weeks) may vary according to the level of antibody in the flock.

OtHer siAdenOviruses

Frog adenovirus 1 is the prototype virus of the genus Siadenovirus. Genetically related viruses have been iden-tified in the tissues of dead raptors (raptor adenovirus 1), and from budgerigars with fatal systemic disease. A siad-enovirus also was recently isolated from Sulawesi tortoises (Indotestudo forsteni) that exhibited anorexia, lethargy, oral erosions, diarrhea, and nasal and ocular discharge. Virus was detected by PCR assay, and isolated from plasma and tissue samples.

oThEr AdEnovirusEsAlthough adenoviruses have been identified in the tissues of several species of fish, including cod, dab, Japanese eel, and bream, to date a virus has been isolated only from white sturgeon (Acipenser transmontanus). Genetic analyses indi-cate that this virus is distinct from those in the other four recognized genera within the Adenoviridae, thus it is pro-posed that this virus serve as the prototype of a new (fifth) genus, Ichtoadenovirus.