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

Table 1. Ocular Adenoviruses

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

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).

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.

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 α_vβ₃ and α_vβ₅, 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 lysosome^34,35 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.

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.

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

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.

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.

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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