Chapter 96
Adenovirus
SANJOY K. GUPTA and WILLIAM G. HODGE
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OVERVIEW
CLASSIFICATION OF ADENOVIRUS
MOLECULAR BIOLOGY OF ADENOVIRUS
PATHOGENESIS
DIAGNOSIS
PREVENTION AND TREATMENT
ADENOVIRUS-MEDIATED GENE TRANSFER
SUMMARY
REFERENCES

OVERVIEW
In the 1950s a novel group of viral agents that were associated with respiratory ailments in humans were discovered. They were named adenoviruses after the adenoids, the original tissue from which the prototypical virus was isolated.1,2 Adenoviruses are a group of double-stranded deoxyribonucleic acid (DNA) viruses that belong to the family Adenoviridae. This family is in turn subdivided into two genera, Aviadenovirus and Mastadenovirus.3 Members of the latter genus infect humans.

There are at present 49 distinct serotypes of adenovirus that cause self-limiting disease in immunocompetent individuals—varied acute infections of the respiratory tract,4 hemorrhagic cystitis,5 and diarrhea.6 Uncommon clinical entities attributable to this virus include meningoencephalitis,7 intussusception,8 and celiac disease.9 There is growing evidence that the virus contributes to the morbidity and mortality of immunocompromised patients.10

Two classic presentations of adenoviral ocular disease are epidemic keratoconjunctivitis (EKC) and pharyngoconjunctival fever (PCF). EKC is most commonly caused by adenoviral serotypes 8, 19, and 37; the latter serotype has predominated since 1977.11 The clinical manifestations of EKC12 include preauricular lymphadenopathy; hyperemia; watery discharge; and acute follicular conjunctivitis, which may be hemorrhagic. Pseudomembranes are found, and subepithelial corneal infiltrates occur in 50% of cases sometimes accounting for prolonged visual loss. The incubation period is 5 to 12 days, and patients may be infectious for up to 2 weeks. Transmission is thought to occur most commonly by direct personal contact.

PCF13 is most commonly caused by adenoviral serotypes 3, 4, 7, and 14. Unlike EKC, concurrent systemic manifestations are common. Although follicles and preauricular lymphadenopathy are common signs of PCF, hemorrhages and pseudomembranes are rare. Corneal signs that are seen in EKC are rare in PCF and, if present, are transitory. Transmission is most commonly believed to occur by droplet spread or from contaminated swimming pools and ponds.

Most cases of adenoviral infection today have a mixed presentation. Indeed at least nine other serotypes have been isolated from ocular sources, thereby explaining why most cases cannot be easily pigeon-holed into a diagnosis of EKC or PCF.14

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CLASSIFICATION OF ADENOVIRUS
There are 49 distinct serotypes of human adenovirus.15 Serotype classification is based on antigenic determination. All human adenoviral serotypes share a common antigen (alpha) located on the hexon capsomere.16 The type specific antigens are the (e) antigen also located on the hexon and (gamma) reactivity located on the capsid fiber.17 Table 1 summarizes the known ocular adenoviral serotypes, their original source of isolation, and clinical diagnosis.15

 

Table 1. Ocular Adenoviruses


TypeSourceClinical Diagnosis
3Nasal washingCommon cold
4Throat washingPneumonia
7Throat washingPharyngitis
8Eye swabEKC
10Eye swabConjunctivitis
14Throat swabURTI
15Eye swabConjunctivitis
16Eye swabConjunctivitis
17Eye swabConjunctivitis
19Eye swabTrachoma
20Eye swabTrachoma
21Eye swabTrachoma
22Eye swabTrachoma
23Eye swabTrachoma
24Eye swabTrachoma
37Eye swabEKC

EKC, epidemic keratoconjunctivitis; URTI, upper respiratory tract infection.
(Horwitz MS: Adenoviruses. In Fields BN, Knipe DM et al [eds]. Fields Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996:2149–2171)

 

Subclassification of the virus is based on several properties. The first subclassification was based on hemagglutination with monkey and rat red blood cells. This subdivided the virus into four main groups.18 The oncogenic potential of the virus in rodents is another subclassification, which mimics closely the hemagglutination groupings.19 The most confusing subgrouping is composed of the tumor (T) antigens shared within a group. The definition of these antigens has undergone considerable evolution and is still has not reached a consensus.20 DNA restriction endonucleases have allowed for a final subclassification scheme based on DNA sequence heterogeneity.21 A summary of adenoviral classification schemes is presented in Table 2.22

 

Table 2. A Summary Classification System for Human Adenoviruses


SubgroupHemagglutination Transformation Groups in Tissue CultureSerotypes %G + CTumors in Animals
AIV (little + to none)12, 18, 31High
   48, 49 
BI (complete of + monkey RBCs)3, 7, 11, 14Moderate
  50–52 
  16, 21, 34, 35 
CIII (partial of + rat RBCs)1, 2, 5, 6Low
  57–59 
DII (complete of + rat RBCs)8, 9, 10, 57–61Low
  13, 15, 17, 19, 20, 22–30, 32, 33, 36–39 
  42 
EIII (complete of low rat RBCs)4-
  57–59 
FIII40, 41-

RBCs, red blood cells.
(Shenk T: Adenoviridae: The viruses and their replication. In Fields BN, Knipe DM et al [eds]. Fields Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996:2111–2148)

 

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MOLECULAR BIOLOGY OF ADENOVIRUS

GENOME STRUCTURE AND GENE EXPRESSION

The 49 serotypes of human adenovirus have been subclassified into six subgroups (A through F).15 The human adenovirus serotypes 2 and 5 (Ad2 and Ad5, respectively), both from subgroup C, have been most extensively characterized and have been used recently for construction of viral vectors for gene therapy. (See the following section on adenovirus-mediated gene transfer.) Ad2 and Ad5 genomes have been completely sequenced and are 95% identical at the nucleotide level, with a similar organization of transcriptional units (reviewed in ref 22). The Ad5 genome is a linear double-stranded DNA of approximately 36 kilobases (kb), with at least 30 different messenger ribonucleic acids (mRNAs). The mRNAs may be grouped into early and late transcripts, according to whether they are expressed before or after viral DNA replication (Fig. 1). Early transcripts (E1A, E1B, E3, and E4) encode proteins that modulate the host cell cycle, mediate viral DNA replication, and control the host immune response to viral invasion.23 Late transcripts (termed L1 through L5) encode viral structural proteins such as hexon, penton base, and fiber. In the late phase of the viral cycle, the major late promoter (MLP) is activated and a large precursor mRNA is synthesized. The L1 to L5 transcripts are derived from this common transcript through alternative splicing. Other small transcripts that have important functions in the viral life cycle encode pIX (a structural protein), IVa2 (a protein that activates the MLP), and VA RNA I and II (RNAs that block activation of the host interferon response).

Fig. 1. Transcription map and genome organization of adenovirus and adenovirus-derived gene transfer vectors. A schematic representation of the adenovirus genome is shown at the top of the figure. The length of the linear double-stranded adenovirus genome is approximately 36 kb and is arbitrarily divided into 100 map units. Black boxes at the terminal ends of the genome represent inverted terminal repeats (ITR). Arrows indicate the direction of transcription for the major messenger ribonucleic acids (mRNAs). E1–E4 indicate early transcripts, L1–L5 represent late transcripts, and MLP is the position of the major late promoter. Below are represented the first-, second- and third-generation (or “gutted”) adenovirus vectors. The transcription regions that are deleted are indicated in each vector. (Adapted from Wu Q, Moyana T, Xiang J: Cancer gene therapy by adenovirus-mediated gene transfer. Curr Gene Ther 1:101, 2001)

STRUCTURE OF THE VIRUS

Adenoviruses are nonenveloped viruses. The viral structural proteins are DNA-associated or involved in formation of the complex capsid encapsulating the viral DNA. The DNA-associated proteins include the terminal protein (TP) that binds to the 5' termini of the viral genome containing the inverted terminal repeats (ITRs).24 Other core proteins include polypeptides V and VII and Protein X. Like cellular histones, these proteins are rich in the basic amino acid arginine, but unlike histones they contain appreciable amounts of tryptophan.25,26 The precise function of each of the core proteins has yet to be elucidated, but protein V may play a role in interfering with host cellular function by redistributing nucleolar antigens.27

The icosahedral capsid structure of adenovirus is made up primarily of three proteins: hexon, penton base, and fiber as shown in Figure 2. The capsid is formed from 252 unit structures, known as capsomeres, arranged in an icosahedral structure with 20 triangular sides. Hexagonal capsomeres or hexons comprise 240 of the structural subunits.28 Each hexon is composed of three different polypeptides VI, VII, and IX. There are also 12 pentagonal capsomeres or pentons. Each penton is composed of an 85 kilodalton (kDa) protein, polypeptide III. Polypeptide III is the largest protein of the entire viral capsid. Projecting from each penton is a rodlike structure called a fiber made up of three molecules of Protein IV, a 62-kDa polypeptide.29 The fiber and penton base not only contribute to the capsid structure but are intimately involved in the attachment of the viral particle to the host cell surface.

Fig. 2. Structure of adenovirus. The icosahedral capsid structure of adenovirus is made up of three proteins, hexon, penton base, and fiber. A cross-sectional view of the viral capsid. Some proteins are associated with viral DNA, whereas others are associated with hexon and are involved in the formation of the capsid.

VIRAL LIFE CYCLE

The replication cycle of adenovirus may be divided into three stages, host infection, DNA replication, and construction of new viral particles. The fiber protein of the viral capsid attaches to a primary receptor on the host cell-surface, much like an anchor. Three types of host cell surface molecules can act as the primary receptor: the coxsackievirus group B and adenovirus receptor (CAR),30 the major histocompatibility complex (MHC)-I α2 subunit,31 and sialic acid residues on glycoprotein.32 Once the viral particle is tethered through the primary interaction, a second interaction between the penton base and a secondary cell-surface receptor, the integrins αvβ3 and αvβ5, take place. The integrins, in turn, promote internalization of the viral particle through receptor-mediated endocytosis.33 Once inside the cell, the outer capsid protein is partially disassembled because of acidification of the contents of the endosome. This allows the virus to escape the endosome before the formation of the lysosome34,35 and enter the cytoplasm. The process of viral internalization and release to the cytosol takes about 15 minutes.36 Virus particles are then translocated to the nucleus along the microtubule network.37 The adenoviral particle undergoes further disassembly, and, as a final step, the Ad hexon proteins remain at the nuclear membrane while the DNA is released into the nucleus.36

The stage of viral synthesis has been divided into early and late phases, the latter beginning after DNA replication.15 Host cell RNA polymerase II is the enzyme that transcribes all viral mRNA.37 Early viral mRNA synthesis has been subdivided into immediate early, delayed early, and intermediate transcription. Immediate early gene products act to regulate all subsequent early transcription.38 In addition, there is a domain of the E1A region that controls the initiation of DNA synthesis. The earliest viral protein to be expressed is E1A, which in turn induces the synthesis of other early proteins, E1B, E2, E3, and E4. Region E1B codes three polypeptides, the smallest of which, a 19-kDa protein, is the most essential.39

Region E2A codes for a single-stranded DNA binding protein of 72 kDa made in abundance in viral infected cells.40 The E2B region encodes two polypeptides, one of which is the precursor of the TP already mentioned.41 There is good evidence that the E3 region plays an important role in modulating the host response to adenoviral infection.42 The products of this region are dispensable for growth in tissue culture. However, one of its proteins, a 19-kDa glycosylated polypeptide binds to the MHC polypeptide heavy chain intracellularly and prevents it from being expressed on the cell surface. Two other proteins from the E3 region are also immunomodulatory in their function. One is instrumental in inhibiting the lysis of adenovirus-infected cells by tumor necrosis factor. The other has been shown to bind to the epidermal growth factor receptor. The E4 region produces many viral proteins, most of which have accessory roles in the viral replicative cycle.43

The intermediate transcripts code for two proteins, IX and IVa2, both of which are essential for virion assembly. Although both are synthesized early, they continue to be synthesized during and after completion of DNA synthesis. In fact, their rate of production in the late phase far exceeds that in the early phase of the replicative cycle.44

Synthesis of adenoviral DNA begins the late phase of the replicative cycle. On viral DNA synthesis, host DNA synthesis is almost completely shut down.45 Each DNA strand elongates by a continuous mechanism in the 5' to 3' direction with the pro TP molecule serving as the initial primer for DNA replication.46 Several nuclear factors have been isolated that are important in DNA replication. Nuclear factor I is a host cell protein that binds near the origin of adenoviral DNA and is essential for initiation and elongation of viral DNA synthesis.47 Nuclear factor II is a 30-kDa complex of two polypeptides48 and is necessary for full elongation of adenoviral DNA. Nuclear factor III recognizes the octamer sequence ATGCAAAT located between 36 and 54 nucleotides from the adenoviral origin of replication.49 The final factor needed for replication is termed ORP A and binds within the first 12 nucleotides of the viral genome.50 During the late phase of the replicative cycle, late mRNA synthesis begins,51,52 and host cell protein synthesis has completely shut down.53 Thus, study of viral transcription during this period has been greatly facilitated. Many of the genes transcribed during this portion of the replicative cycle code for the structural proteins already described.54 The events that control the switch from early to late viral gene expression have yet to be clearly elucidated. One hypothesis is that TP modification is instrumental in this process.55

Virion assembly is the final stage of the replicative process. Assembly begins when single polypeptides are assembled into capsomeres in the cytoplasm. Hexon monomers are assembled quickly (in only a few minutes).56 The pentons are synthesized with biphasic kinetics, one fourth are assembled rapidly in approximately 20 minutes, the rest take more than 10 hours. The isolation of temperature-sensitive mutants has led to the discovery of a 100-kDa scaffold protein extremely important in the assembly process.57 This protein is necessary for the assembly of the virion but is absent in the final product.

With the aid of temperature-sensitive mutants, several stages of virion assembly have been elucidated. The next recognizable stage of virion assembly is the light intermediate capsid stage.58 This stage consists of assembly of a structural intermediate consisting of the hexon, as well as proteins IIIa, VI, and VIII. Two proteins, 39-and 50-kDa, serve as scaffold proteins in the formation of this intermediate. A small piece of DNA from the left end of the viral genome is also associated with this intermediate.59 Next, viral DNA enters the preformed capsids through one of its vertices, and the core polypeptides are added.56

Viral DNA replication begins approximately 7 hours after infection, after which the late proteins are expressed. Viral particle assembly occurs 20 to 24 hours after infection, and approximately 104 virions per cell are released in 2 to 3 days with lysis of the host cell.

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PATHOGENESIS
Adenoviral infection disrupts normal cellular physiology at many different levels. The disruption of host cell DNA synthesis, as well as normal transcription, is incompatible with cell survival.60 Moreover, the viral penton is directly toxic to host epithelial cells.61 These events lead to epithelial cell death and lysis. Lytic infection results in the production of about 1 million progeny viruses per cell, 5% of which are infectious.60 It is this lytic cycle that is responsible for most symptoms and signs associated with adenoviral conjunctivitis. Adenovirus may also establish a latent infection, which most commonly occurs in lymphoid tissue.62 However, latent infection has also been demonstrated in monkey epithelial cells. Whether latent infection might contribute to the generation of subepithelial corneal opacities seen with adenoviral keratitis remains speculative.

An important part of the pathogenesis of adenoviral ocular disease is the molecular epidemiology of infection. Despite group-specific and type-specific antigens, adenoviral conjunctivitis continues to occur often in epidemic outbreaks. Mutations in other areas of the adenoviral genome allow the virus to evade the immune system of its host to establish infection.

Two studies have added to the understanding in this area. Itakura and colleagues63 studied the subtypes of adenovirus type 4 occurring during EKC outbreaks in Sapporo, Japan between 1985 and 1989. During this period, 122 strains were isolated, and by restriction endonuclease analysis it was shown that the prevalent genotypes changed from outbreak to outbreak within several years. In a second study, de Jong and coworkers64 studied four consecutive outbreaks of EKC secondary to serotype 8 over a 5-year period in Brest, France. By restriction endonuclease analysis, they found no genomic variation within an epidemic but large changes between epidemics. The plasticity of the adenoviral genome in these strains undoubtedly plays a role in the formation of new outbreaks that violate the immunosurveillance of individuals within the epidemics.

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DIAGNOSIS
Diagnosis of adenoviral infection is often based on clinical grounds alone. However, if the diagnosis is in doubt or precise documentation of infection is required, various assays are available. Common to all of these is the collection of specimens into a suitable transport medium that will stabilize the virus and inhibit overgrowth of bacteria and fungi. A typical transport medium consists of gelatin-based tissue culture media containing the antibiotics penicillin, streptomycin, and amphotericin. If immediate inoculation is not possible, the virus may be stored at -70°C.65

Viral culture is the gold standard method of laboratory diagnosis. Human epithelial cell lines are the best hosts for viral isolation and replication. Adenoviral infection of cultured cell lines may be detected by the presence of the “cytopathogenic effect.”65 This consists of characteristic rounding and swelling of infected cells typically starting at the periphery of the monolayer. However, adenovirus-infected cells also increase the rate of glycolysis and formation of lactic acid. Hence, pH indicators may also be useful in identification of adenovirus-infected cells.66

A simple way to diagnose adenoviral conjunctivitis is by conjunctival cytology. Intranuclear inclusions are the light microscopic benchmark of adenoviral nuclear assembly. Unfortunately recognition of these inclusions requires experience, and other viral infections including herpes simplex may produce similar inclusions. Hence, this technique lacks both the sensitivity and specificity of viral culture. Other diagnostic methods67 such as fluorescent antibody techniques and complement fixation lack the sensitivity of culture or have yet to be tested in a rigorous clinical fashion.

Polymerase chain reaction (PCR)-based diagnostic techniques for the detection and classification of adenovirus are being developed and may soon provide a rapid alternative to the gold standard of viral culture. Using PCR primers specific to the fiber protein, a rapid multiplex PCR assay correctly identified the subgroups of all 49 known serotypes, as well as 180 field isolates.68 All six subgroups were distinguished in another described PCR assay based on the hexon protein coding region; in addition, the PCR assay could differentiate between serotypes 8, 19, and 37 that cause ocular disease.69 Another exciting PCR assay investigation described identification of nine oculopathogenic serotypes of subgroup D.70 This assay detected positive Ad serotypes in 48 out of 102 samples (47%), whereas culture isolation and neutralization assays yielded only 29 of 102 positive results, suggesting a greater sensitivity of PCR. Moreover, the results of the PCR assay and the culture assay with regard to the serotype were 100% concordant.70

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PREVENTION AND TREATMENT
The most important aspect of managing adenoviral ocular disease is preventing its spread in epidemic proportions. Usually this simply requires examining a patient in an isolated “red eye” room that is vigorously cleaned with dilute bleach. In severe epidemics more extreme actions must be taken. In a large outbreak of EKC at The Illinois Eye and Ear Infirmary in the mid 1980s, more than 400 patients were infected in a 6-month period.71 Measures used to halt the transmission of the virus included examining all patients entering the building for infection, establishing isolation waiting rooms for those suspecting of having the infection, dispensing all medication in unit dose form, and assigning staff whose sole function was to take care of these patients. Furthermore, health employees suspected of being infected were removed from work for 14 days.

For the infected individual, little can be offered except education on limiting spread and measures for comfort. The latter includes cool compresses, cycloplegia, and avoidance of ocular medication with preservatives. For the rare patient with persistent corneal subepithelial infiltrates, topical corticosteroids can have a dramatically beneficial result.

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ADENOVIRUS-MEDIATED GENE TRANSFER
The aim of successful gene therapy is to introduce a foreign gene into the tissue of interest and to achieve long-term expression of the transgene. Because of the ability of adenoviruses to infect a wide variety of cell types in a cell-cycle independent fashion, adenovirus-based vectors have been receiving much attention in the past decade.

The wild-type adenovirus is capable of accepting a small amount of foreign DNA (2 kb). Foreign DNA that is of greater length, when introduced into adenovirus, results in viral instability. Early approaches to the use of adenovirus for gene transfer, thus, focused on the problem of deletion of viral genomic DNA to accommodate larger sizes of foreign DNA. In the past 7 years, three generations of adenoviral vectors have been developed; these are shown schematically in Figure 1.

First-generation adenoviral vectors were constructed by deletion of the E1 and/or E3 regions.72 The purpose of deletion of E1 sequences was threefold: to abolish the potential oncogenicity of the adenovirus vectors (E1A and E1B have transforming activity in culture), to make the vector replication deficient, and to increase the cloning capacity of viral vector (for review, see ref 73). The major drawback of the first-generation vectors was their tendency to elicit robust immune responses, both to the viral capsid proteins and to infected cells expressing viral antigens. The early immune response was against capsid proteins and mediated by proinflammatory cytokines and inflammatory cells. The later T-cell response was to the foreign gene product and to viral antigens that are produced at low levels in infected host cells even with the absence of E1 gene product. Moreover, transgene expression with adenoviral vectors was typically transient, lasting 1 to 2 weeks, and future vector readministrations were rendered ineffective because of strong immune responses following primary exposure to the vector.

Second-generation vectors attempted to remove (in addition to E1) other elements required for viral replication, including E2 or E4 coding regions.74–77 These vectors required propagation in cell lines that provided the complementary function for viral replication (i.e., E1 and E2 or E4). The results from second-generation vector gene transfer experiments were mixed, with no to marked improvement in long-term transgene expression and reduced immunogenicity and toxicity compared with the first-generation vectors. An interesting observation was that Ad vectors with deletion of E4 sequences showed reduced transgene expression over time, which appeared to be through cell-mediated downregulation of promotors (such as the cytomegalovirus immediate-early promoter) used to drive the transgene.78,79 Subsequently, a polypeptide in the E4 region, E4ORF3, was identified; its presence prevented downregulation of viral promoters. Inclusion of E4ORF3 in subsequent second-generation vectors resulted in a improved duration of transgene expression.80

Recently, third-generation vectors have been developed that lack the entire viral genomic sequence, except for the ITRs at each end and the packaging signal (Ψ) that is required to package viral DNA into viral particles. These are known as fully deleted Ad vectors (fdAd) or “gutted” vectors (reviewed in ref 81). These are capable of accepting up to 37 kb of foreign DNA for gene transfer. These vectors are propagated in the presence of helper virus, which provides all replicative functions in trans. Ideally, one would like to obtain a virus extract with minimal contaminating helper virus, because the presence of helper virus will accentuate host immune responses. A helper virus that lacks the packaging signal would not be able to package itself but only the fdAd construct that contains the packaging signal. However, such a helper virus is hard to propagate. Elegant mechanisms have been developed with inherent “suicide” signals in the helper virus. A currently used technique is shown in Figure 3, where the helper virus packaging signal is flanked by loxP sites.82 The loxP site is a target for the bacteriophage P1 Cre recombinase. In the presence of active Cre recombinase, the two loxP sites undergo recombination, causing excision of the helper virus Ψ signal. Such a helper virus can be easily propagated in normal cell lines. Co-infection of the fdAd vector construct containing the Ψ signal and the helper virus in a cell line expressing Cre recombinase allows the fdAd construct to be packaged, whereas the loxP recombination prevents the helper virus from being packaged. This system allows the contamination of helper virus to be reduced to as low as 0.01% of the fdAd titer.

Fig. 3. Propagation of gutted adenoviral vector constructs. This diagram is a schema for the propagation of gutted vectors. The helper phage is E1 deleted and must be propagated in a cell line (293) that expresses E1. The helper phage contains a packaging signal (Ψ) that is flanked by loxP recombination target sites. The gutted vector construct contains the flanking inverted terminal repeats, the foreign DNA that is driven by a viral promoter, and a stuffer fragment. The 293Cre cell line expresses the bacteriophage P1 Cre recombinase. When the 293Cre cell line is concomitantly infected with the helper phage and the gutted vector construct, the helper phage packages the gutted vector construct. Meanwhile, the helper phage undergoes loss of the packaging signal (Ψ) through recombination of the loxP sites with Cre recombinase and cannot be packaged. This process allows amplification and packaging of the gutted vector construct by serial passage, while minimizing helper phage contamination of the prepared virus stock. (Adapted from Parks RJ, Chen L, Anton M et al: A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A 93:13565, 1996)

Ad vectors have been used in gene therapy protocols thus far for the destruction of diseased cells or the long-term restoration of defective or missing genes. Ad vectors have been particularly promising in cancer gene therapy (reviewed in ref 73). One therapeutic approach is to allow for delivery of cytokines that have anti-oncogenic activity. The delivery of tumor suppressor genes such as p53 to trigger apoptosis has been successful in treatment of prostate, breast, and thyroid cancer. A third approach has been the adenovirus-mediated delivery of chemogenes, that is, enzymes that can convert prodrugs at the tumor site and allow the drug to work at the site of action, while reducing toxic side effects of the chemotherapeutic agent to normal tissue.

A number of major problems still face this new treatment modality.73,83 First, adenoviral constructs are unable to infect tumor cells that lack the primary cell receptors such as CAR. Second, the presence of primary receptors for adenovirus on normal cells of many tissues results in infection of normal bystander cells and also in a lack of tissue-specificity. For the correction of defective or absent genes, the long-term expression of Ad vector-mediated transgenes remains a problem. Efforts are underway to overcome these problems.

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SUMMARY
The spectrum of human adenoviral infection is wide, and morbidity is significant. This virus is one of the most common pathogens of the external eye. In this chapter, we have discussed the structure and physiology of the virus, the clinical manifestations it produces, and the rationale for preventive therapeutic action. An exciting new dimension in the field of adenovirus virology is the development of gene transfer methods using adenoviral vectors. We have attempted to provide the reader with a bird's eye view to this emerging new field of therapeutics.
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REFERENCES

1. Hilleman MR, Werner JH: Recovery of new agents from patients with acute respiratory illness. Proc Soc Exp Biol Med 85:183, 1954

2. Rowe WP, Huebner RJ, Gilmore LK et al: Isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proc Soc Exp Biol Med 84:570, 1953

3. Norrby E, Bartha A, Boulanger P et al: Adenoviridae. Intervirology 7:117, 1976

4. Brandt CD, Kim HW, Vargosdo AJ et al: Infections in 18,000 infants and children in a controlled study of respiratory tract disease. I. Adenovirus pathogenicity in relation to serologic type and illness syndrome. Am J Epidemiol 90:484, 1969

5. Namazaki Y, Kumasaka T, Yano N et al: Further study of acute hemorrhagic cystitis due to adenovirus type 11. N Engl J Med 289:344, 1973

6. Moffett HL, Shulenberger HK, Burkholder ER: Epidemiology and etiology of severe infantile diarrhoea. Pediatrics 72:1, 1968

7. Chou SM, Roo R, Burrell R et al: Subacute focal adenovirus encephalitis. J Neuropathol Exp Neurol 32:34, 1973

8. Bell TM, Steyn JH: Viruses in lymph nodes of children with mesenteric adenitis and intussusception. Br Med J 2:700, 1962

9. Kagnoff MF, Peterson VJ, Kumar PJ et al: Evidence for the role of a human intestinal adenovirus in the pathogenesis of coeliac disease. Gut 28:995, 1987

10. Zahradnik JM, Spencer MJ, Parker DD: Adenovirus infection in the immunocompromised patient. Am J Med 68:725, 1980

11. Kemp MC, Hierholzer JC, Cabradilla CP et al: The changing etiology of epidemic keratoconjunctivitis antigenic and restriction enzyme analysis of adenovirus types 19 and 37 isolated over a 10 year period. J Infect Dis 148:29, 1983

12. Jawetz E, Thygeson P, Hanna L et al: The etiology of epidemic keratoconjunctivitis. Am J Ophthalmol 43:S79, 1957

13. Bell JA, Rowe WP, Engler JI et al: Pharyngoconjunctival fever. Epidemiological studies of a recently recognized disease entity. JAMA 175:1083, 1955

14. Bennett FM, Law BB, Hamilton W et al: Adenovirus eye infections in Aberdeen. Lancet 2:670, 1957

15. Horwitz MS: Adenoviruses. In Fields BN, Knipe DM et al (eds). Fields Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996:2149–2171

16. Norrby E: The relationship between soluble antigens and the virion of adenovirus type 3. I. Morphological characteristics. Virology 28:236, 1966

17. Philipson L, Pettersson U, Lindberg U: Molecular biology of adenoviruses. Virol Monogr 14:1, 1975

18. Rosen I: A hemagglutination-inhibition technique for typing adenoviruses. Am J Hyg 71:120, 1960.

19. Huebner RJ, Casey MJ, Chanock RM et al: Tumours induced in hamsters by a strain of adenovirus type 3. Sharing of tumour antigens and “neoantigens” with those produced by adenovirus type 7 tumours. Proc Natl Acad Sci U S A 54:381, 1965

20. McAllister RM, Nicholson MO, Reed G et al: Transformation of rodent cells by adenovirus 19 and other group D adenoviruses. J Natl Cancer Inst 43:917, 1969

21. Pina M, Green M: Biochemical studies on adenovirus multiplication. IX. Chemical and base composition analysis of 28 human adenoviruses. Proc Natl Acad Sci U S A 54:547, 1965

22. Shenk T: Adenoviridae: The viruses and their replication. In Fields BN, Knipe DM et al (eds). Fields Virology. 3rd ed. Philadelphia: Lippincott-Raven, 1996:2111–2148

23. Flint J, Shenk T: Viral transactivating proteins. Annu Rev Genet 31:177, 1997

24. Rekosh DM, Russell WC, Bellet AJ et al: Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283, 1977

25. Laver WG, Suriano JR, Green M: Adenovirus proteins. II. N-terminal amino acid analysis. J Virol 1:723, 1967

26. Hosakawa K, Sung MT: Isolation and characterization of an extremely basic protein from adenovirus type 5. J Virol 17:924, 1976

27. Matthews DA: Adenovirus protein V induces redistribution of nucleolin and B23 form nucleolus to cytoplasm. J Virol 75:1031, 2001

28. Everitt E, Sundquist B, Pettersson U et al: Structural proteins of adenoviruses. X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2. Virology 62:130, 1973

29. van Oostrum J, Burnett RM: The molecular composition of the adenovirus type 2 virion. J Virol 56:439, 1985

30. Bergelson JM, Cunningham JA, Droguett G et al: Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320, 1997

31. Hong SS, Karayan L, Tournier J et al: Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J 16:2294, 1997

32. Arnberg N, Edlund K, Kidd AH et al: Adenovirus 37 uses sialic acid as a cellular receptor. J Virol 74:42, 2000

33. Wickham TJ, Mathias P, Cheresh DA et al: Integrins alpha v beta 3 and alpha v beta5 promote adenovirus internalization but not virus attachment. Cell 73:309, 1993

34. Mellman I: The importance of being acidic: The role of acidification in intracellular membrane traffic. J Exp Biol 172:39, 1992

35. Leopold PL, Ferris B, Grinberg I et al: Fluorescent virions: Dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells. Hum Gene Ther 9:367, 1998

36. Greber UF, Willetts M, Webster P et al: Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75:477, 1993

37. Berk AJ, Sharp PA: Sizing and mapping of early adenovirus mRNAs by gel electrophoresis os S1 endonuclease digested hybrids. Cell 12:721, 1977

38. Moran E, Mathews MB: Multiple functional domains in the adenovirus E1A gene. Cell 48:177, 1987

39. Matsuo T, Wold WSM, Hashimoto S et al: Polypeptides encoded by transforming region E1B of human adenovirus 2: Immunoprecipitation from transformed and infected cells and cell-free translation of E1B-specific mRNA. Virology 118:456, 1982

40. Linne T, Jornvall H, Philipson L: Purification and characterization of the phosphorylated DNA binding protein from adenovirus type 2 infected cells. Eur J Biochem 76:481, 1976

41. Friefeld BR, Lichy J, Hurwitz J: Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149. Proc Natl Acad Sci U S A 80:1589, 1983

42. Burgert HG, Maryanska JL, Kvist S: E3/19k protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell-surface expression of histocompatability class I antigens. Proc Natl Acad Sci U S A 84:1356, 1987

43. Sarnow P, Hearing P, Anderson CW: Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix. J Mol Biol 162:563, 1982

44. Shaw AR, Ziff EB: Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomyosin block of protein synthesis. Mol Cell Biochem 2:789, 1982

45. Pina M, Green M: Biochemical studies on adenovirus multiplication. XIV. Macromolecule and enzyme synthesis in cells replicating oncogenic and non-oncogenic human adenoviruses. Virology 38:573, 1969

46. Lechner RL, Kelly TJ Jr: The structure of replicating adenovirus 2 DNA molecules. Cell 12:1007, 1977

47. Diffley JF, Stillman B: Purification of a cellular double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter binding assay. Mol Cell Biol 6:1363, 1986

48. Guggenheimer RA, Nagata K, Field J et al: In vitro synthesis of full length adenoviral DNA. In Cozzarelli N (ed). UCLA symposia on mechanisms of DNA replication and recombination. Vol 10. New York: Alan R. Liss, 1983:395–421

49. Pruijn GJ, van Driel W, van der Vliet PC: Nuclear factor III, a novel sequence-specific DNA binding protein from HeLa cells stimulating adenovirus DNA replication. Nature 322:656, 1986

50. Rosenfeld PJ, O'Neill EA, Wides RJ et al: Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication. Mol Cell Biol 7:875, 1987

51. Evans R, Weber J, Ziff E et al: Premature termination during adenovirus transcription. Nature 278:367, 1979

52. Zeevi M, Nevins JR, Darnell JE Jr: Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid. Mol Cell Biol 2:517, 1982

53. Anderson CW, Baum PR, Gesteland RF: Processing of adenovirus 2 induced proteins. J Virol 12:241, 1973

54. Hosakawa K, Sung MT: Isolation and characterization of an extremely basic protein from adenovirus type 5. J Virol 17:924, 1976

55. Thomas GP, Mathews MB: DNA replication and the early to late transition in adenovirus infection. Cell 22:523, 1980

56. Horwitz MS, Scharff MD, Maizel JV Jr: Synthesis and assembly of adenovirus 2. I. Polypeptide synthesis, assembly of capsomeres and morphogenesis of the virion. Virology 39:682, 1969

57. Lundholm U, Doerfler W: Temperature sensitive mutants of human adenovirus type 12. Virology 45:827, 1971

58. D'Halluin JC, Milleville M, Boulanger PA et al: Temperature sensitive mutant of adenovirus type 2 blocked in virion assembly: Accumulation of light intermediate particles. J Virol 26:344, 1978

59. Hammarskjold ML, Winberg G: Encapsidation of adenovirus 16 DNA is directed by a small DNA sequence at the left end of the genome. Cell 20:787, 1980

60. Ginsberg HS: Adenoviruses. In Davis BD, Dulbecco R, Eisen HN et al (eds). Microbiology. Hagerstown, MD: Harper & Row, 1980:1047

61. Pettersson U, Hoglund S: Structural proteins of adenoviruses. Virology 39:90, 1969

62. Abken H, Butzler C, Willecke K: Adenovirus type 5 persisting in human lymphocytes is unlikely to be involved in immortalization of lymphoid cells by fusion with cytoplasts or by transfection with DNA of mouse L cells. Anticancer Res 7:553, 1987

63. Itakura S, Koki A, Harumi S et al: Changes in subgenome types of adenovirus type 4 isolated from patients with ocular disease between 1985 and 1989 in Sapporo, Japan. J Clin Micro 29:1740, 1991

64. de Jong JC, Demazure M, Legrand-Quillien MC: New developments in the molecular epidemiology of adenovirus 8 keratoconjunctivitis. J Med Virol 38:102, 1992

65. Kasel JA. Adenoviruses. In Lennette E, Schmidt N (eds). Viral, Rickettsial and Chlamydial Infections. New York: American Public Health Association, 1980:114–142

66. Fisher TN, Ginsberg HS: Accumulation of organic acids by HeLa cells infected with type 4 adenovirus. Proc Soc Exp Biol Med 95:47, 1957

67. Gardner PS, McQuillin J: Rapid diagnosis application of immunofluorescence. London: Butterworth, 1974:181–193

68. Xu W, McDonough MC, Erdman DD: Species-specific identification of human adenoviruses by a multiplex PCR assay. J Clin Microbiol 38:4114, 2000

69. Elnifro EM, Cooper RJ, Klapper PE et al: PCR and restriction endonuclease analysis for rapid identification of human adenovirus subgenera. J Clin Microbiol 38:2055, 2000

70. Adhikary AK, Numaga J, Kaburaki T et al: Rapid detection and typing of oculopathogenic strain of subgenus D adenoviruses by fiber-based PCR and restriction enzyme analysis. Invest Ophthalmol Vis Sci 42:2010, 2001

71. Warren D, Nelson KE, Farrar JA: A large outbreak of epidemic keratoconjunctivitis: Problems in controlling nosocomial spread. J Infect Dis 160:938, 1989

72. Graham FL, Prevec L: Manipulation of adenovirus vectors. In Murray EJ, Clifton NJ (eds). Methods in Molecular Biology. New York: Humana, 1984:109–128

73. Wu Q, Moyana T, Xiang J: Cancer gene therapy by adenovirus-mediated gene transfer. Curr Gene Ther 1:101, 2001

74. Lusky M, Christ M, Rittner K et al: In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. J Virol 72:2022, 1998

75. Fang B, Wang H, Gordon G et al: Lack of persistence of E1-recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs. Gene Ther 3:217, 1996

76. Amalfitano A, McVie-Wylie AJ, Hu H et al: Systemic correction of the muscle disorder glycogen storage disease type II and hepatic targeting of a modified adenovirus vector encoding human acid-alpha-glucosidase. Proc Natl Acad Sci U S A 96:8861, 1999

77. Hu H, Serra D, Amalfitano A: Persistence of an [E1-, polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice. Hum Gene Ther 10:355, 1999

78. Brough DE, Hsu C, Kulesa VA et al. Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo. J Virol 71:9206, 1997

79. Armentano D, Zabner J, Sacks C et al: Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J Virol 71:2408, 1997

80. Yew NS, Marshall J, Przybylska M et al: Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3. Hum Gene Ther 10:1833, 1999

81. Parks RJ: Improvements in adenoviral vector technology: Overcoming barriers for gene therapy. Clin Genet 58:1, 2000

82. Parks RJ, Chen L, Anton M et al: A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A 93:13565, 1996

83. Breyer B, Jiang W, Cheng H et al: Adenoviral vector-mediated gene transfer for human gene therapy. Curr Gene Ther 1:149, 2001

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