263Fenner and White’s Medical Virology. DOI:© 2012 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-375156-0.00018-72017

In 1953 Wallace Rowe and his colleagues observed that certain explant cultures of human adenoids degenerated spontaneously. On prolonged culture, they isolated a new virus they referred to as adenovirus. It quickly became evident not only that many adenoviruses persist for many years as latent infections of lymphoid tissues, but also many are a significant cause of respiratory and ocular disease. Later studies also implicated related viruses as the cause of genitourinary tract infections, and still later adenoviruses were found associated with gastroenteritis and with infections of immunocompromised patients. Following the discovery that certain human adenoviruses produce malignant tumors in neonatal rodents, molecular biologists turned their attention to the biochemistry and molecular biology of adenovirus replication and the events leading to oncogenesis. Although adenoviruses were eventually shown to play no role in human cancer, the spin-off from this research has had a major impact on our understanding of the expression of mammalian as well as viral genes. One landmark contribution was the discovery of RNA transcript splicing, for which Philip Sharp and Richard Roberts were awarded the Nobel Prize in Physiology or Medicine in 1993.

Adenoviruses cause 5% to 10% of all febrile illnesses in infants and young children. Most individuals have serological evidence of prior adenoviral infection by the age of 10. Adenovirus infections are especially prevalent in daycare centers and in households with young children. Many epidemics of adenoviral disease have been described, including pharyngoconjunctival fever in summer camps and public swimming pools, keratoconjunctivitis in medical facilities, and serious acute respiratory disease in military recruits.

Adenoviruses have been used as vectors for recombinant-DNA vaccines and for gene therapy due to the very high yield of adenoviruses in cell cultures coupled with a lack of oncogenic properties in humans.

CLASSIFICATION

The family Adenoviridae consists of five genera. Human adenoviruses together with other adenoviruses infecting mammals belong to the genus Mastadenovirus, whereas

adenoviruses of birds, sheep, cattle, frogs, and fish are grouped into the separate genera Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus.

The classification of human adenoviruses is in transition, switching from a system based upon classical serological methods to genome sequencing methods. Serotypes had been distinguished by hemagglutination and neutralization assays which involve antigens on virion fibers and hexons. Human adenoviruses 1 to 52 were classified in this way. The discovery and classification of human adenoviruses types 52 to 68 have been based on genome sequencing and bioinformatic analysis. Biological properties, lack of cross-neutralization, sequence relatedness, pathology, and other properties have been used to group these human viruses into seven species (human adenoviruses A to G), each of which is associated with a distinct disease-association profile, including a varying capacity to induce tumors in experimental animals (Table 18.1). The species human adenovirus-D contains the most members, including a substantial number identified during the first two decades of the AIDS epidemic. Adenovirus epidemiology keeps evolving: in recent years, one virus, human adenovirus 14, has been associated with severe, even fatal outbreaks of pneumonia in residential facilities and

Chapter 18

Adenoviruses

TABLE 18.1 Classification of Human Adenoviruses

Subgroup Serotypes Tropism Production of Tumors in Animals

A 12, 18, 31 Intestinal High

B1 3, 7, 14, 16, 21, 50 Respiratory Moderate

B2 11, 14, 34, 35 Renal Moderate

C 1, 2, 5, 6 Respiratory Low or none

D 8 to 10, 13, 15, 17, 19, 20, 22 to 30, 32, 33, 36 to 39, 42 to 49, 51, 53, 54

Ocular and other

Low or none

E 4 Respiratory Low or none

F 40, 41 Intestinal Low or none

264 PART | II Specific Virus Diseases of Humans

military bases. Homologous recombination in nature appears to play a major role in generating genome diversity.

PROPERTIES OF ADENOVIRUSES

Adenovirus virions are non-enveloped, with a 70 to 90 nm diameter capsid that is a perfect icosahedron. Structural studies of adenoviruses have focused on adenovirus serotypes 2 and 5. The capsid is composed of 252 capsomers: 240

hexons constitute the 20 equilateral triangular facets, while 12 pentons are located over the 12 vertices. A fiber protrudes from each penton to give adenoviruses one of the most distinctive morphological appearances among all viruses (Fig. 18.1). The distal ends of the fibers bear ligands for cellular receptors and determine the host species specificity of the virus. The genome, which is associated with an inner protein core, consists of a single linear molecule of dsDNA, 26 to 48 kbp in size, with inverted terminal repeats. The

FIGURE 18.1 (A) Negative contrast electron microscopy of human adenovirus 5—clinical respiratory diagnostic specimen. (B) Single adenovirus virion showing its surface architecture. (C) Thin-section electron microscopy of human adenovirus 5 in human embryonic kidney cell culture, showing a typical paracrystalline array of virions in the nucleus of an infected cell. (D, left) Model of an adenovirus virion showing the virion surface composed of hexons and pentons—penton fibers are located at each vertex of the icosahedral virion. (D, right) Diagrammatic section of a virion showing the location of the major proteins. The structure of the nucleoprotein core has not been established so the location of core proteins is hypothetical. (D, right) Reproduced from Flint S.J., et al., 2009. Principles of Virology: Pathogenesis and Control, third ed. ASM Press, Washington, DC, with permission.

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DNA, in association with a 55 K protein which is covalently linked to each 5′ terminus, is infectious when transfected into susceptible cells.

There are about 11 viral structural proteins, among which protein II is the hexon, protein III the penton, protein IV the fiber, and a 23 kDa cysteine protease is responsible for processing other viral precursor proteins. Fibers of the majority of human adenoviruses comprise only one protein, but human adenoviruses 40 and 41 have two distinct fibers, of different lengths and primary sequence, present in the virion in equimolar amounts; this may extend the host ranges of these serotypes. The other minor proteins have been shown either to stabilize the hexons under different chemical environments, to participate in disrupting the endosomal membrane in viral invasion, or to assist in assembly of the virion. Polypeptide VII is the major core protein of the virus, while the minor core protein V functions to attach the core to the capsid.

Due to possessing a dsDNA genome, compact icosahedral capsid and the lack of a lipid envelope, adenoviruses are relatively resistant to the environment, consistent with recorded transmission in swimming pools. The virus is stable between pH 6.0 and 9.5, which aids the transmission via the fecal–oral route. Adenoviruses in simulated conjunctival samples can be shipped at ambient temperatures without loss of titer. Adenoviruses are resistant to chloroform, ether, and fluorocarbons. Recent studies recommend disinfecting ophthalmology equipment with 70% ethyl alcohol or approximately 5000 parts per million chlorine. When adenoviruses are heated to 56°C, the virus disintegrates, and the core is released (Table 18.2).

VIRUS REPLICATION

Most adenoviruses are species-specific and generally will undergo a complete replication cycle only in cells derived from the native host species. In common with many DNA viruses, adenoviruses replicate within the cell nucleus where nascent virions are also assembled.

The virion enters cells by attachment of fiber “knobs” to a primary docking protein, for example, to the Coxsackie adenovirus receptor (CAR), or to CD46 or CD80, after which viral binding and entry are facilitated by the interaction between the RGD motif in the penton base and different cell surface integrins. Recent studies have shown that human adenovirus 5 (HAdV5) can attach to dendritic cells via a bridging mechanism involving the binding of lactoferrin to the C-type lectin receptor DC-SIGN. This may not only affect the host range of human adenovirus 5 in cells, but also has implications for using adenoviruses in gene delivery. Interestingly, human adenovirus 41, a cause of gastroenteritis, has two fibers, with only one attaching to CAR: the second is thought to attach to enterocytes. After attachment, internalization occurs through the interaction of the penton base and cellular integrins via clathrin-coated vesicles. The complex is then transferred into endosomes, where the viral capsid is partially degraded by a virus-coded protease, and viral DNA transported into the nucleus. Within the nucleus, viral DNA becomes attached to the nucleus matrix via its terminal protein, followed by the well-regulated transcription of early and late mRNAs.

Transcription of adenovirus mRNAs follows a sequential pattern, divisible into early and late stages, closely paralleling a similar pattern of expression among the well-characterized DNA bacteriophages. The viral genome contains five early transcription units (E1A, E1B, E2, E3, and E4), three delayed early transcription units (IX, IVa2, and E2 late), and one late transcription unit (major late), the latter processed to generate five families of late mRNAs (L1 to L5). All mRNAs are transcribed by cellular RNA polymerase II. Each of the early units has its own separate promoter, while the major late unit uses just a single promoter. Each of these units gives rise to multiple mRNAs that are created by alternative splicing, thereby making use of the limited genome sequences to translate a large number of proteins. Multiple splicing of the early transcripts leads to the production of around 30 mRNAs, coding for early proteins that are mainly involved in viral replication.

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 to provide an optimal environment for DNA synthesis and viral replication, (2) protection of infected cells from host antiviral immune defenses, such as from tumor necrosis factor (TNF) activity or from apoptosis, and (3) synthesis of viral proteins necessary for viral DNA replication. E1A and E1B gene products are also responsible for cell transformation and

TABLE 18.2 Properties of Adenoviruses

Five genera; Mastadenovirus includes 68 human serotypes grouped into 7 genera designated A to G, and serotypes that infect other mammals. Genus and type-specific classification is mainly based on serological assays, supplemented by phylogenetic analysis

Non-enveloped virion 70 to 90 nm in diameter, hexagonal outline, icosahedral symmetry, with 240 hexons, 12 pentons with 12 fibers that mediate attachment to receptors

Contain a linear double-stranded DNA genome, 26 to 48 kb, with inverted terminal repeats and a protein primer at each 5′ end

Transcription, DNA replication, and assembly occur in the cell nucleus

Complex sequential program of early, intermediate, and late mRNA transcription (before and after DNA replication); extensive splicing of RNA transcripts

Some viruses are oncogenic in laboratory animals, but none have been associated with human cancer

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FIGURE 18.2 Adenovirus genome organization (human adenovirus 2). The genome of adenoviruses consists of a single linear molecule of double-stranded DNA, 26 to 48 kbp in size, with inverted terminal repeats. The genome encodes approximately 40 proteins that are transcribed by cellular RNA polymerase II according to a complex program involving both DNA strands and complex RNA splicing. Different virion proteins are transcribed in different directions (arrows). There are early and late transcriptional units, each under the control of different promoters. Viral DNA replication proceeds from both ends by a strand-displacement mechanism. ITR, inverted terminal repeat; TP, terminal protein; ML, major late tripartite leader. Reproduced from Flint, S.J., et al., 2009. Principles of Virology: Pathogenesis and Control, third ed. ASM Press, Washington, DC, with permission.

hence for the animal oncogenicity of some adenoviruses. Both proteins interact with the cellular tumor suppressor gene p53 to compromise its normal cellular function and thus allow cell cycle progression. Proteins translated from E2 are directly involved in viral DNA replication, and include a DNA polymerase, an ssDNA binding protein, and a precursor to the terminal protein. The E3 region is not essential for adenovirus replication in cell cultures and can be deleted or replaced without disrupting viral replication in vitro. It is therefore favored as an insertion site for foreign 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 expression by infected cells and inhibition of tumor necrosis factor are two examples of immune evasion mediated by E3 encoded proteins. Studies of E4 mutants have shown that adenovirus inhibits the cellular DNA damage response.

Multiple splicing of the late genes gives rise to at least 18 mRNAs that code for the L1 to L5 families of late proteins which are involved in the assembly of progeny virions. The phenomenon of RNA splicing was discovered owing to the very high abundance of late mRNAs processed during adenovirus transcription. In addition to the above, adenoviruses contain two VA (viral associated) genes (VAI and VAII), which are transcribed by host RNA polymerase III to produce short VA RNA molecules that mimic the function of dsRNA due to the presence of two stem loop structures. These VA RNAs are not translated but are essential for lytic virus replication, and can inhibit both the host interferon system and host RNAi (Figs. 18.2 and 18.3).

Viral DNA synthesis is initiated at the end of inverted terminal repeats at each end of the genome, using the 5′-linked virus-coded precursor protein primer to supply

the priming function normally provided by the 3′OH of an upstream DNA strand. DNA synthesis then proceeds from both ends of the genome by asymmetric strand synthesis that involves copying one strand and displacing the other. The elongation of viral DNA requires the participation of adenovirus DNA polymerase, ssDNA binding protein and a cellular topoisomerase. The displaced single-stranded DNA molecule forms a panhandle-like structure by annealing of its term inal repeat sequences, which then act as the site of origin for replication of the complementary DNA strand, finally giving rise to a full-length double-stranded progeny molecule.

Following DNA replication, late mRNAs are transcribed and translated into structural proteins. The shutdown of host cell macromolecular synthesis occurs progressively during the later stages of the replication cycle. The assembly of progeny virus occurs within the nucleus. During assembly, the viral protease cleaves at least four viral products in order to make the released virion more stable and thus enhance infectivity. Many thousands of assembled virions can be observed in the infected cell nucleus, arranged into a paracrystalline array. The release of progeny virions is aided by an increase in the permeability of the nuclear membrane mediated by a viral non-structural protein. The release of progeny virions is dependent upon cell lysis. In cell cultures, many adenoviruses induce severe condensation and margination of the host cell chromatin, making the nuclei appear abnormal, and appearing as the inclusion bodies so characteristically observed in adenovirus-infected cells.

Abortive adenovirus infection can occur with the expression of early genes, when the combined actions of E1A and E1B can induce cell transformation or tumor formation in animal models. There are also situations where adenoviruses persist in vivo and will remain as life-long infections that may lead to life-threatening disease in an immunocompromised host.

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ADENOVIRUS DISEASES

Clinical Features

Only about half of the known human adenoviruses have been causally linked to disease (Table 18.3). Adenoviruses 1 to 8 are much the commonest species worldwide and are responsible for most cases of adenovirus-induced disease. Some 5–10% of acute respiratory illnesses in children under the age of five years (but less than 1% of those in adults) have been ascribed to adenoviruses. Enteric human adeno viruses, human adenoviruses 40 and 41, have been claimed to cause up to 10% of infantile gastroenteritis. Several common adenoviruses, including adenoviruses 8, 19, and 37 are major causes of eye infections and also cause genital infections.

Respiratory Infections

Acute respiratory infections are seen particularly in young children and are clinically similar to infections caused by other respiratory viruses. The infant presents with a cough, nasal congestion, and fever; the throat is inflamed

and there is often an exudative tonsillitis that resembles group A streptococcal infection. Adenoviruses 1 to 7 are usually responsible for these common sporadic infections that are relatively trivial except when otitis media or pneumonia supervene. Acute respiratory disease occurs in epidemic form when military recruits assemble in camps. Adenoviruses 4 and 7 are most often responsible.

Pneumonia, often severe and occasionally fatal, may develop in young children infected with any of the common adenoviruses, but particularly human adenoviruses 7 and 3. In some of the colder parts of the world, such as northern China and Canada, adenoviruses are an important cause of pneumonia in infants under the age of two. Adenovirus pneumonia may be associated with disseminated infection involving the heart, liver, kidney, pancreas and CNS with a fatality rate of 10% to 30%, and many of the survivors show permanent lung damage. Acute respiratory disease in military recruits also occasionally progresses to a pneumonitis. Severe infections, including pneumonia, seen in immunosuppressed patients are of increasing importance in this era of organ transplantation and AIDS.

FIGURE 18.3 Replication cycle of adenoviruses (human adenovirus type C). See text for details. Reproduced from Viral Zone, Swiss Institute of Bioinformatics, with permission.

268 PART | II Specific Virus Diseases of Humans

Ocular Infections

Pharyngoconjunctival fever tends to occur in outbreaks, for example, at childrens’ summer camps (“swimming pool conjunctivitis”), and is associated with types 3 and 7. Adenovirus 4 has caused a number of nosocomial outbreaks of conjunctivitis or pharyngoconjunctival fever among hospital staff.

Epidemic keratoconjunctivitis is a more severe eye infection, commencing as a follicular conjunctivitis and progressing to involve the cornea (keratitis). The disease once known as “shipyard eye,” is highly contagious and often occurs as epidemics caused by adenoviruses 8, 19, and 37. Adenovirus 8 has been the principal cause, but in 1976 adenovirus 37 suddenly appeared, spread worldwide, and today is the predominant cause of epidemic keratoconjunctivitis.

Genitourinary Infections

Cervicitis and urethritis are common manifestations of venereal infection with adenovirus 37, first identified in prostitutes. Hemorrhagic cystitis, seen mainly in young boys, is caused by adenovirus 11 and more rarely adenovirus

21. Adenoviruses commonly establish asymptomatic persistent infection of the kidney and may be shed in the urine for months or years. This is observed particularly in immunocompromised individuals, such as renal transplant recipients.

Enteric Infections

Gastroenteritis in infants is commonly caused by adeno-viruses 40 and 41. These enteric adenoviruses, previously visualized by electron microscopy in feces and long regarded as difficult to isolate, can now be grown in cultured cells. The recovery of these viruses from outbreaks of gastroenteritis, and recovery significantly more frequently from sympto-matic patients than from controls, confirms that human adenoviruses do indeed cause gastrointestinal disease. However, many other adenoviruses that replicate in the intestine or in the throat are excreted asymptomatically in the feces for weeks or months, hence carefully controlled studies are required before assigning these viruses an etiological role in gastroenteritis.

Adenoviruses have also been suspected or suggested to be involved in other diseases such as myocarditis,

TABLE 18.3 Diseases Caused by Human Adenoviruses

Disease Age Common Serotypesa Major Subgenus Major Source

Respiratory Infections

Pharyngitis Young children 1, 2, 3, 5, 6, 7 B, C Throat

Acute respiratory disease Military recruits 3, 4, 7, 14, 21 B, E Throat

Pneumonia Young children 1, 2, 3, 4, 5, 7, 21 B, C Throat

Military recruits 4, 7 B, E Throat

Ocular Infections

Pharyngoconjunctival fever Children 1, 2, 3, 4, 6, 7 B, C, E Throat, eye

Epidemic keratoconjunctivitis Any age 8, 19, 37 D Eye

Genitourinary Infections

Cervicitis, urethritis Adults 19, 37 D Genital secretions

Hemorrhagic cystitis Young children 11, 21 B Urine

Enteric Infections

Gastroenteritis Young children 31, 40, 41 A, F Feces

Infections in Immunocompromised Individuals

Encephalitis, pneumonia Any age, including AIDS patients

7, 11, 34, 35 B Urine, lung

Gastroenteritis AIDS patients Many D including 43 to 47 D Feces

Generalized AIDS patients 2, 5 C Blood

aOnly the commonly occurring serotypes are listed; those most commonly associated with particular syndromes are in bold type.

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cardiomyopathy, meningoencephalitis, and hepatitis, because of the recovery of virus (or detection by PCR) from series of patients with these diseases.

Infections in Immunocompromised Patients

Adenoviruses have emerged as an important cause of life-threatening infections in at least three groups of immunocompromised patients. In children with severe combined immune deficiency disease common adenoviruses can cause serious conditions such as pneumonia or meningoencephalitis. Transplant recipients, particularly children following stem cell transplantation, and patients with AIDS, often shed subgroup B adenoviruses (11, 34, and 35) in their urine for prolonged periods, and may develop high fever, pneumonia, hemorrhagic cystitis, encephalitis, hepatitis, or nephritis. AIDS patients also may excrete adenoviruses 43 to 47 in their feces.

PATHOGENESIS, PATHOLOGY, AND IMMUNITY

Adenoviruses are widespread in many animal species and can be readily isolated from healthy individuals, persisting life-long in lymphoid tissues. Adenoviruses multiply initially in the pharynx, conjunctiva, or small intestine, and generally do not spread beyond the draining cervical, pre-auricular, or mesenteric lymph nodes. Usually, the disease process remains relatively localized, and the incub-ation period is short (five to eight days). Most enteric infections and some respiratory infections are subclinical. Generalized infections are occasionally seen, especially in immunocompromised patients, and also in those undergoing transplantation. Deaths do occur, particularly from adenovirus 7, the most virulent human adenovirus. At autopsy, lungs, brain, kidney, liver, and other organs reveal the characteristic basophilic nuclear inclusions referred to above. One possible molecular mechanism that may enhance dissemination of virus is the release of excess viral penton fiber protein from infected cells at cell lysis. The fiber protein binds the epithelial junction protein desmoglein 2 (DSG2), thereby triggering intracellular signaling and transient opening of the junctions between epithelial cells. This may facilitate lateral spread and dissemination of virus in epithelial tissues.

Infection with the common endemic types 1, 2, and 5 can persist asymptomatically for years in the tonsils and adenoids of children, with virus continuously shed in the feces for many months after the initial infection, then intermittently for years thereafter. The mechanism of this persistence is uncertain; perhaps viral replication is held in check by the antibody synthesized by these lymphoid organs. Fluctuation in shedding indicates that latent adenovirus infections can be reactivated. For example, this can occur

during infection with Bordetella pertussis, and measles can be followed by adenovirus pneumonia. Most adenovirus infections are localized in the eyes and pharynx, but in some cases there is contiguous extension into the lungs. Although most adenoviruses replicate harmlessly in the intestine, human adenoviruses 40 and 41 can cause gastroenteritis. Some adenoviruses are frequently shed in the urine of immunocompromised persons; adenoviruses 34 and 35 were originally isolated from renal transplant recipients and can be recovered commonly from AIDS patients. As there is no evidence of ascending infections, urinary bladder infection suggests that the virus probably is viremic at some stage in order to reach this organ. In addition, a wide variety of other adenoviruses have been recovered from the feces of AIDS patients. Many of these viruses are new or rare and some appear to be genetic recombinants. Presumably these novel viruses and “intermediate” strains arose in this cohort as a result of mixed infections and the greater levels of replication resulting from immunosuppression. Infections caused by other adenoviruses are characterized by prolonged latency in lymphoid tissue, but can also be reactivated in AIDS patients and frequently recovered from the blood.

Outbreaks of adenovirus infections can be caused by new adenovirus variants with a high virulence for patients without an immunodeficiency. For example, community based outbreaks of severe respiratory disease occurred at three different locations in USA in 2006–2007, caused by a human adenovirus 14 variant. Of 140 patients with acute Ad14 respiratory disease, 38% required hospitalization, 17% needed admission to an intensive care unit, and there were 9 deaths. Therefore the continuous monitoring of adenovirus infections is advised together with genetic analysis of viral strains over time in order to forecast the risk of an outbreak in the community.

In contrast to most other respiratory viral infections, adenovirus infections lead to lasting immunity to reinfection with the same serotype, perhaps because of the extent of involvement with lymphoid cells in the alimentary tract and the regional lymph nodes. Maternal antibody generally protects infants under the age of 6 months against severe lower respiratory disease. Relatively little is known of cell-mediated immune responses against adenoviruses in humans. CD4+ and CD8+ T cell epitopes exist in the conserved regions of the hexon protein and these epitopes are cross-reactive among some adenovirus species. However it is not known whether or not these T cell epitopes are protective against adenovirus. The virus can also modulate host T cell responses by expression of several early gene products. For example, one small protein encoded by a gene within the E3 transcription unit binds to the heavy chain of the class I MHC antigen, and prevents transport of class I MHC to the cell surface, thereby decreasing the presentation of adenovirus peptides to cytotoxic T lymphocytes. Another E3 gene-product protects infected cells against lysis by

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tumor necrosis factor, whereas other products stimulate clearance and degradation of the receptors for Fas ligand, TRAIL and epidermal growth factor from the cell surface, thereby interfering with intracellular signaling by these ligands. Due to the increasing use of adenoviruses for gene delivery, the nature of immune responses against adenoviruses is of particular interest in assessing the efficacy of heterologous gene expression from adenovirus vectors. For example, the longstanding solid immunity following infection is a problem in the use of certain serotypes as vectors in immune individuals.

LABORATORY DIAGNOSIS

Depending on clinical presentation, appropriate specimens include feces, pharyngeal swabs, nasopharyngeal aspirates, transtracheal aspirates, or bronchial lavage. Eye infections can be diagnosed by the taking of conjunctival swabs, corneal scraping, or tears. Other syndromes may involve sampling genital secretions, urine, biopsy (e.g., of liver or spleen) or autopsy (e.g., lung or brain) samples.

Enzyme immunoassay (EIA) is the method of choice for the detection of soluble viral antigen in feces or nasopharyngeal secretions. A monoclonal antibody to a hexon epitope common to all adenoviruses or polyclonal serum suffices to identify the family, then if desired, a type-specific mono clonal antibody can be used to identify the particular adenovirus concerned. Rapid point-of-care immunochromato graphic tests have also shown good sensitivity and specificity. Immunofluorescence can also be employed to demonstrate adenoviral antigen in cells from the respiratory tract, eye, urine, or biopsy or autopsy material, after low-speed centrifugation of the specimen followed by fixation of the pelleted cells; however it tends to have lower sensitivity than EIA, particularly among adults.

The most sensitive method for virus detection is polymerase chain reaction (PCR) using genus-specific primer sets. This can be incorporated into a multiplex assay to detect a panel of common respiratory pathogens, and is also useful to quantitate the adenovirus DNA load, as a high load is more often associated with active disease. PCR can also be used for environmental detection of viruses, for example, in wastewater, surface water, and combined sewer overflows.

Virus isolation is still an approach used by some diagnostic and reference laboratories. Cell culture of adenoviruses is time-consuming because many of the viruses are very slow-growing. Human malignant cell lines such as HeLa, HEp-2, KB, or A-549, or diploid human embryonic fibroblasts are the substrates of choice. The fastidious enteric adenoviruses 40 and 41 have only recently been cultivated in vitro and require special cell lines such as Graham-293 which express the human adenovirus 5 E1A and E1B genes,

or special conditions, for example, the use of a low-serum cell medium. The common adenoviruses (adenoviruses 1 to 7) generally produce cytopathology within one to two weeks; the cells become swollen, rounded, and refractile, cluster together like a bunch of grapes, and reveal characteristic basophilic intranuclear inclusions. Appropriate type-specific antisera, or monoclonal antibodies directed to type-specific epitopes on the fiber, can then be chosen to type the isolate by HI and/or neutralization.

EPIDEMIOLOGY, PREVENTION, CONTROL, TREATMENT

Adenoviruses are mainly associated with disease of the respiratory tract and eye. Virus is often transmitted by respiratory droplets or contact, particularly in outbreaks of pharyngoconjunctival fever in children (adenoviruses 3, 4, and 7), or acute respiratory disease in military recruits (adenoviruses 4 and 7) in late winter and spring. Types 1, 2, 5, and 6 are mostly associated with sporadic respiratory infections. A live-attenuated virus vaccine for human adenoviruses 4 and 7 has been used to prevent respiratory adenovirus infections in military recruitment camps in the United States, but such a vaccine has not been extended to young children.

Eye infections may be acquired by transfer of respiratory secretions on the fingers. Two important settings for direct entry to the eye are “swimming pool conjunctivitis” where the chlorination of water has been inadequate, and nosocomial infection—a number of major outbreaks of epidemic keratoconjunctivitis have been traced to the surgeries of particular ophthalmologists or hospitals where aseptic technique is inadequate.

Spread occurs also by the enteric (fecal–oral) route, with very large numbers of adenovirus particles shed into feces (1011 virions per gram) usually for 1 to 14 days. However, shedding can last for several months, thus representing a continuing source of infection. Adenoviruses 40 and 41 are endemic worldwide and often cause asymptomatic infection, but they have been clearly demonstrated to cause gastroenteritis in children the year around, with occasional outbreaks, for example, in schools or hospitals.

Adenovirus can be excreted in urine, while adenoviruses 19 and 37 can presumably be transmitted venereally as well as by contact in eye infections because they can cause genital ulcers and urethritis in both sexes.

A number of antiviral drugs including ribavirin, ganciclovir, and cidofovir have shown variable in vitro activity against adenoviruses, but in clinical use ribavirin has shown little efficacy. Cidofovir may be of some benefit, and it is sometimes used together with intravenous immunoglobulin for the treatment of severe infections.

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Prevention of infection relies on careful attention to respiratory isolation particularly during outbreaks, handwashing, proper disinfection of ophthalmic equipment, adequate chlorination of swimming pools, and similar measures to prevent spread.

Beginning in 1971, military recruits in the United States received a highly effective, enteric-coated, oral live vaccine with adenovirus types 4 and 7; manufacture of this vaccine was discontinued in 1996. The incidence of adenovirus respiratory disease, including pneumonia, increased substantially, so the vaccine program was reinstituted. In 2011, a new live, oral adenovirus vaccine against adenovirus serotypes 4 and 7 was approved for use in the United States military personnel aged 17 to 50 years of age.

ADENOVIRUSES AS VECTORS FOR THE DELIVERY OF HETEROLOGOUS DNA

Adenoviruses offer considerable advantages for gene delivery based on the following points: (1) virus stocks can be reliably propagated to high levels, (2) the lack of integration into host chromosomes means that the chance of insertional oncogenesis is negligible, and the duration of the expression of the transgene will be limited, especially in cells with rapid turnover, (3) diseases caused by adenoviruses are mostly mild or subclinical, (4) adenovirus vaccines have been used in military populations, and there is experience regarding the immune responses induced by this virus. In addition, genetic manipulation of the virulence genes can further attenuate adenoviruses, while engineering of receptor-binding domains can be done to promote infection of specific cell targets, making the delivery of foreign DNA more effective.

Adenoviruses as vectors can be classified into two main categories, replication-defective and replication-competent. Replication-defective vectors serve as an inert vehicle to deliver the transgene into the target cell, whereas for replication-competent vectors virus replication in the target cell is part of the intended mechanism of action. In the first generation of replication-defective vectors, E1A and E1B regions were deleted and replaced by the insertion of the transgene. In addition the non-essential E3 region was also deleted to increase cloning capacity. After further refinements, replication-defective vectors are now being used for several therapeutic purposes. These include the delivery of genes to control cell growth and apoptosis, for the treatment of cancer through administration of cytotoxic genes. In separate studies, adenoviruses are being used to prevent the overgrowth of the arterial wall during the healing phase after angioplasty for the opening of blocked cardiac arteries.

A further use of adenovirus vectors is to deliver DNA expressing an epitope or antigen as a vaccine. Both the

humoral and cell-mediated immune responses can be stimulated by these approaches. Antigens that have been inserted into adenoviruses include the hepatitis B surface or core antigens, HIV-1 env, gag, or p24 proteins, pseudorabies virus gD protein, Epstein-Barr virus glycoprotein 340/220, vesicular stomatitis virus structural glycoprotein, rotavirus VP4, rabies virus glycoprotein, bovine parainfluenza virus 3 glycoprotein, and feline immunodeficiency virus envelope glycoprotein.

One important obstacle that limits the in vivo use of adenovirus as a vector is the high prevalence of neutralizing antibodies against human adenoviruses 5, which has been most commonly used as gene therapy vector. Pre-existing antibodies or antibodies generated by repeated systemic administration of human adenovirus 5, will neutralize the vector, resulting in failure of the therapy. To circumvent this, researchers have been developing vectors based on other less common serotypes, for instance human adenovirus 48 or non-human adenoviruses that are mostly resistant to neutralizing antibodies against human adenovirus 5.

Replication-competent vectors may be more promising for the treatment of cancers. Oncolytic adenovirus vectors kill cancer cells as part of the natural adenovirus life cycle, so following replication the virions are released from the lysed tumor cell to infect surrounding cells within the tumor.

Despite the promise of adenoviruses for gene therapy, one significant failure was the HIV-1 vaccine STEP trial, which was halted after two years in 2007 because individuals seropositive for adenovirus 5 showed increased rates of HIV-1 acquisition after vaccination with a human adenovirus 5 vaccine. It was shown that using the adenovirus-based vaccine in individuals with pre-existing immunity against human adenovirus 5 resulted in the preferential expansion of HIV-susceptible activated CD4+ T cells homing to mucosal tissues, thereby increasing the number of virus targets. This led to a greater susceptibility to acquiring HIV. This setback demonstrates the complexity of using adenovirus for gene delivery and gene immunization in clinical trials. More work is needed to fully reveal the appropriate use of adenoviruses in gene therapy and gene immunization.

FURTHER READING

Hoeben, R.C., Uil, T.G., 2013. Adenovirus DNA replication. Cold Spring Harb. Perspect. Biol. 5, a013003.

Majhen, D., Calderon, H., Chandra, N., et  al., 2014. Adenovirus-based vaccines for fighting infectious diseases and cancer: progress in the field. Human Gene. Ther. 25, 301–317.

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