Chapter 18 Adenoviruses 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 cevident 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% 10 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 fare grouped into the separate genera Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus. The classification of human adenoviruses isin 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 vition fibers and hexons. Human adenoviruses 11052 were classified inthis way. The discovery and classification of human adenoviruses types $2 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, hhuman adenovirus 14, has been associated with severe, even fatal outbreaks of pneumonia in residential facilities and TABLE 18.1 Classification of Human Adenoviruses. Subgroup | Serotypes Tropism | Production ‘of Tumors in Animals a 12,1831 Intestinal [High a 3,7, 14,16, 21,50 | Respiratory | Moderate 2 11,14,34.35 [Renal Moderate © 12,56 Respiratory [Low or none D ‘81010, 13, 15,17, [Ocularand [Low or none 19,20,22%030, ° | other 32,33, 362039, 2 049, 51,53, 54 E 4 Respiratory [Low or none F 40,44 Intestinal [Low or none 263264 PART | II Specific Virus Diseases of Humans Viral DNA FIGURE 18.1. (A) Negative contrast elston microscopy of fvman adenovirus S—ctinical respiratory diagnostic specimen. (B) Single enoviros ‘von showing its suTae architecture. (C) Thin Section election microscopy of human adenovirus Sia human embryonic Kidaey cel elt, showing 4 ‘ypial paracrine aray of virion nthe nucleus of an infec cell (D let) Model ofan adenovirus virion showing the virion surface composed of. exons and pentons—peston fibers ae located at each vertex ofthe iosahedal virion. (D ght Dagranimti section of a virion showing the locaton of the major proteins, Te stractze of the miclenproten core has ot been estblisied so the location of core protein ix bypathesa. (, right) Reproduced ‘from Flint Sa, 200 Principles of Vnoogy: Pathogenesis and Cont third d. ASM Pres, Washington, DC with permision. rilitary bases. Homologous recombination in nature appears ‘o play a major role in generating genome diversity. PROPERTIES OF ADENOVIRUSES Adenovirus virions are non-enveloped, with a 70 to 90nm diameter capsid that is a perfect icosahedron, Structural studies ofadenoviruseshave focused on adenovirusserotypes 2 and 5. The capsid is composed of 252 capsomers: 240 hhexons constitute the 20 equilateral triangular facets, while 12pentons 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 48kbp in size, with inverted terminal repeats. TheDNA, in association witha 55K 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 ITs the hexon, protein III the penton, protein IV the fiber, and a 23kDa 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 distinet 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 ofthe virus, while the minor core protein V functions to attach the core to the capsid, Duetopossessing adsDNA genome, compacticosahedral 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 {© chloroform, ether, and Muorocarbons. 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). TABLE 18.2 Properties of Adenoviruses Five genera; Mastadenovirus includes 68 human seronypes rouped into 7 genera designated Ato G, and serotypes that Infect ther mammals. Genus and type-specic classification is mainly based on serological assays, supplemented by phylogenetic analysis 'Non-enveloped virion 70 to 90nm in diameter, hexagonal putin, icosahedral symmetry, with 240 hexons, 12 pentons with 12 fibers that mediate stachment to receptors Contain a linear double stranded DNA genome, 26 0 4845, with inverted terminal repeats and a protein primer at each Send “ransrition, DNA replication, and assembly occur in the ell nuceus ‘Compiex sequential program of early intermediate, and late mRNA anscrntion before and after DNA replication); extensive spicing of RNA transcripts Some viruses are oncogene in laboratory animals, but none have been associated with human cancer Adenoviruses Chapter | 18 265 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 here 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), of to CD46 or CD80, after which viral binding and entry arefacilitatedby theinteraction between the RGD motif in the penton base and different cell surface integrins. Recent studies have shown that human adenovirus 5 (HAAVS) can attach to dendritic cells via a bridging ‘mechanism involving the binding of lactoferrin o the C-rype lectin receptor DC-SIGN. This may not only affeet the host range of human adenovitus Sin 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 \ell-tegulated 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 wwell-characterized DNA bacteriophages. The viral genome contains five early transcription units (EIA, EIB, E2, E3, and F4), three delayed early transcription units (IX, IVa2, and F2 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 Il. 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 transeripts leads to the production of around 30 mRNAs, coding for eatly proteins that are mainly involved in viral replication, ‘The EIA region ofthe 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. EIA and EIB gene products are also responsible for cell transformation and266 PART | II Specific Virus Diseases of Humans 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 eycle progression. Proteins translated from E2 are directly involved in viral DNA replication, and include a DNA polymerase, an ssDNA binding protein, and 4 precursor to the terminal protein, The F3 region is not essential for adenovirus replication in cell cultures and can be deleted or replaced without disrupting viral replication in vitro. Iti 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 LS 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 (VAT and VAID, which are transcribed by host RNA polymerase IIE 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 inked virus-coded precursor protein primer to supply Penton Cove Proteins Hexon 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-standed DNA ‘molecule forms a panhandle-like structure by annealing of its, terminal repeat sequences, which then act as the site of origin for replication of the complementary DNA strand, finally giving rise toa 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 ccharacteristically observed in adenovirus-infected cells. ‘Abortive adenovirusinfection can occur with the expression of early genes, when the combined actions of ELA 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. iver r 2 Poymerase tet bbna-ninaing protein (ose) es va BE FIGURE 18.2 _Adenovires genome organization (human adenovirus 2. The genome of adenoviruses consists ofa single linear molecule of doable: sanded DNA, 26: 48 Kp in size, with inverted terial repeats The genome encodes approximately 40 proteins tht are tanerbe by celular RNA Polymerase IT scording to a complex program involving bah DNA strands and complex RNA spicing. Different virion proteins ae transcribed in ‘herent dvections ars). There ae early and le wanscripional units, each under the cong of dflerent promotes. Viel DNA replication proceeds ‘fomboth end by astrand-isplacement mechanism. TR, inser terminal pet: P. terminal presen; MI majr ate trparte lade. Reproduced rom Flint, $1, etal, 2008. Principles of Virology: Pathogenesis and Control. third ed ASM Press, Washington, DC with pemassionAdenoviruses Chapter | 18 267 Intermediate Iramedite cary FIGURE 18.3 Replicuon cycle of adenoviruses (human adenovins type C). See tex for deals. Reproduced from Viral Zone, Swiss Institute of Bioinformatics wit pemisson ADENOVIRUS DISEASES Clinical Features Only about half of the known human adenoviruses have been causally linked (o disease (Table 18.3). Adenoviruses 1 8 are much the commonest species worldwide and are responsible for most cases of adenovirus-induced disease. Some 510% 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 adenoviruses, hhuman 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 ceauses 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 tonsilltis 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 eamps. 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 hear, liver, kidney, pancreas and CNS with 4 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.268 PART | II Specific Virus Diseases of Humans TABLE 18.3 Diseases Caused by Human Adenoviruses Disease Age ‘Common Seratypes* ‘Major Subgenus | Major Source Respiratory Infections Pharyaais Young children 123,567) BC Throat ‘Acute respiratory disease Milaryreceuts | 3,4,7, 14,21 BF Throat Preumonia Young eildren 1,2,3,4,5,7,21 BC Throat Miltaryrecruis [4,7 BE Throat (cular Infections Pharyngoconjunctval fever | Chien 123,467 BGE The Epidemic keratoconjunetvitis [Any age 819,37 D Bye Genitourinary infections Conic, wretitis ‘dul 19,37 D Genital secretions Hemorthage este Young children fe @ Urine Enteric Infections Gastoenterts Young children 31, 40,41 AP Feces Infections in Immunocompromised individuals Encephalitis, pneumonia ‘Any age including | 7, 11,34, 35 8 Urine, lng, AIDS patients Gastoenters AIDS patients Many D including #31047 | D Feces Generalized AIDS patients 2.5 c Blood “ny the commonly occuring sevotypes are sted thse most conmonlyasocated with pacur sydiomes aren bold ype Ocular Infections 21, Adenoviruses commonly establish asymptomatic Pharyngoconjunctival fever tends t© 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 comea (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, persistent infection of the kidney and may be shed in the turine 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, ean 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,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 ‘meningocncephalitis. 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, hemorthagic 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 ccan 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, iver, and other organs reveal the characteristic basophilic nuclear inclusions referred {© 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 iggering intracellular signaling and transient opening of the junctions between epithelial cells, ‘This may facilitate lateral spread and dissemination of virus, in epithelial issues. Infection with the common endemic types 1, 2, and 5 ccan 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 mechanistn of this persistence is uncertain; perhaps viral replication is held in cheek by the antibody synthesized by these lymphoid organs Fluctuation in shedding indicates that latent adenovirus, infections can be reactivated, For example, this ean occur Adenoviruses Chapter | 18 269 dduring 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 ceases 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 reeipients and can bbe recovered commonly from AIDS patients. As there is no evidence of ascending infections, urinary bladder infection suggests that the virus probably is viremie at some stage in border 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 ot 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 eplication 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 tree different locations in USA in 2006-2007, caused by ‘a human adenovirus 14 variant, OF 140 patients with acute AdLé respiratory disease, 38% required hospitalization, 177% 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 stains over time in order to forecast the Fisk of an outbreak in the community Tn contrast to most other respiratory viral infections, adenovirus infections lead to lasting immunity to reinfection with the same serotype, pethaps because of the extent of involvement with lymphoid cells in the alimentary tract tnd 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 inhumans. CD44 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 itis not known whether or not these T cell epitopes are protective against adenovirus, The vius 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 T MHC antigen, and prevents transport of class 1 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270 PART | II Specific Virus Diseases of Humans 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 ean be diagnosed by the taking of conjunctival swabs, corneal seraping, 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 hhexon epitope common 0 all adenoviruses or polyclonal serum suffices to identify the family, then if desired, a type-specific monoclonal antibody can be used to identify the particular adenovirus concerned, Rapid point-of-care immunochromatographie 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, of biopsy oF autopsy material, afler 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-specitic primer sets. This ean be incorporated into a multiplex assay to deteet & 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 ccan also be used for environmental detection of vieuses, 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 vieuses, 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 enterie adenoviruses 40 and 41 have only recently been cultivated in vitro and requice special cell lines such as Graham-293, which express the human adenovirus 5 EIA and EIB genes, orspecial conditions, for example, the use of alow-serum cell, medium, The common adenoviruses (adenoviruses 1 10 7) generally produce eytopathology within one to two weeks; the cells become swollen, rounded, and refractle, cluster together like a bunch of grapes, and reveal characteristic basophilic Appropriate type- specific antibodies directed to type-specific epitopes on the fiber, can then be chosen 10 Iype the isolate by HI and/or neutralization intranuclear inclusions, antisera, or monoclonal EPIDEMIOLOGY, PREVENTION, CONTROL, TREATMENT Adenoviruses are mainly associated with disease of the respiratory tract and eye. Virus is often transmitted by respiratory droplets oF contact, particularly in outbreaks of pharyngoconjunetival 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, se infections may be acquired by transfer of respiratory secretions on the fingers. Two important setings for direet enlry 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 als by the enter (feeal-oral) route, with very large numbers of adenovirus particles shed into Teces (10" virions per gram) usually for 1 to 14 days, However, shedding can last for several months, hus representing a continuing source of infection, Adenoviruses 40 and 41 are endemic worldwide and offen cause asymptomatic infection, but they have been clearly demonstrated to cause gastroenteritis in children the year around, with occasional coubreaks, for example, in schools or hospital. ‘Adenovirus can be excreted in urine, while adenoviruses 19 and 37 can presumably be transmitted venereally as wel as by contact in eye infections because they can cause genital uleers and urethritis i both sexes. ‘A number of antiviral drugs including sibaviin, ganciclovir, and cidofovie have shown variable in vitro activity against adenoviruses, but in clinical use ribavirin has shown little efficacy, Cidofovie may be of some benefit, and it is sometimes used together with intravenous immunoglobulin for the treatment of severe infections.Prevention of infection relies on careful attention {© 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 vaceine 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, anew 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: (I) virus stocks cean 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 tumover, (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 fof receptor-binding domains can be done to promote 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 aetion, In the first generation of replication-defective vectors, EIA 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. Alter 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, forthe treatment of eancer through administration ofeytotoxie genes. In separate studies, adenoviruses are being used to prevent the overgrowth of the arterial wall during the healing phase after angioplasty forthe ‘opening of blocked cardiae arteries, A further use of adenovirus vectors is to deliver DNA. expressing an epitope or antigen as a vaccine, Both the infection of speci Adenoviruses Chapter | 18 271 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-I env, gag, or p24 proteins, pseudorabies virus gD protein, Epstein-Barr virus glycoprotein 340/220, vesicular stomatitis virus structural glycoprotein, rotavirus YP4, 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 veetoris 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 ofthe therapy. To circumvent this, researchers have been developing vectors based on other less common serotypes, for instance human adenovirus 48 ot non-human adenoviruses that are mostly resistant to neutralizing antibodies against human adenovirus 5, Replication-competent vectors may be mote promising for the treatment of cancers. Oncolytic adenovirus vectors Kill cancer cells as part of the natural adenovirus life eycle, so following replication the virions ate released from the lysed tumor cell to infect surrounding cells within the tumor. Despite the promise of adenoviruses for gene therapy, cone significant failure was the HIV-1 vaccine STEP tial, Which was halted after two years in 2007 because individuals seropositive for adenovirus 5 showed increased rates of HIV-| 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 CD44 T cells homing to mucosal Lissues, thereby increasing the number of virus targets ‘This led to a greater susceptibility to acquiring HIV, This setback demonstrates the compleity of using adenovirus for gene delivery and gene immunization in clinical tals. Mote work is needed to fully reveal the appropriate use of adenoviruses in gene therapy and gene immunization, FURTHER READING Hocten, RC, Uil TG, 203, Adsnovis DNA replication, Cold Spring ar, Perspect. Biol, 5, 013008, ‘Matben, D, Calderon, H., Chandra, Net al, 2014, Adenovirus-baed ‘actines for Fighting infoeous diseases and cancer progess inthe Seld, Homan Gee, Ther 25, 301-317 ‘Tebregge, M., Corts, N, 2012, Adenovias an overview for peditie Infectious disease specialists, Pela In. Dis. J. 3, 626-627,