Primary human embryonic kidney (HEK) cells are probably the best host for the entire range of human Ads, but such cells are expensive and may be contaminated with adeno-associated virus. Continuous epithelial lines, such as HEp-2, HeLa, KB, HEK 293, and A549, are also highly sensitive. Ads in monolayer cell culture have a characteristic cytopathic effect (CPE). The cells round up, swell, and detach from the culture surface into grape-like clusters, and the nuclei become enlarged³⁶³ (Fig. 56.1). Eventually the cells lyse, leaving cell debris. This CPE is the result of the infection passing into the “late” stage of infection, when Ad DNA, messenger RNA (mRNA), and proteins are being made in large quantities and virions are assembling in the cell nucleus.

Ads have a double-stranded DNA genome of ∼36,000 base pairs (bp) enclosed by a protein capsid and with no membrane (see Chapter 55). The virion binds to specific receptors on the cell surface and enters the cell by endocytosis, and the genome is transported to the cell nucleus. “Early” genes are expressed, viral DNA replicates, “late” genes are expressed, virions assemble in the cell nucleus, and the cells lyse to release progeny virus (see Chapter 55 and the legend to Fig. 56.10). There are about 20 early genes and 15 late genes. Ads increase glycolysis in continuous cell lines and thereby induce the cells to produce large quantities of acid. Rapid cytopathology can be induced within several hours of inoculating concentrated crude virus preparations and is not related to viral replication; rather, it is caused by the penton base component of the free viral penton capsomere.

Nuclear morphologic changes in infected tissue cells can be used for diagnostic purposes. The nuclear changes include overall enlargement and intranuclear inclusions that initially are Feulgen negative and eosinophilic but become Feulgen positive and basophilic as the infection progresses.⁷⁰ The Feulgen-positive inclusions contain Ads by electron microscopy (EM) (see Fig. 56.2), and it is the DNA within the viruses that
contributes to the Feulgen stain. In addition, there are paracrystalline aggregates that contain viral protein without nucleic acid.⁴⁸⁴

The clinically predominant Ad antigens are the three capsid proteins: hexon, penton base, and fiber. Most early studies indicated that the hexon and to a lesser extent the fiber proteins contain most of the epitopes recognized by neutralizing antibodies, but there are neutralizing epitopes on penton base as well (reviewed in¹⁰⁹,²¹⁸,³⁰²,⁴⁴⁸,⁵⁶⁹,⁵⁹¹,⁶⁹³,⁷⁸⁶). The neutralization properties of polyclonal antibodies are often concordant with the inhibition of Ad-induced hemagglutination (HA) of selected red blood cells. However, the HA functions are the property of fiber (reviewed in⁵²¹), which usually must be linked to the penton base for complete HA to occur. Because of recombination within species in clinical isolates, it is not uncommon to isolate a virus that demonstrates discordant reactions in the neutralization and HA inhibition reactions.

One recent study of naturally infected humans found that the majority of neutralizing antibodies were against the hypervariable regions of hexon, but some were also against fiber and possibly penton base.⁵⁹¹,⁶⁹³ In contrast, another recent study concluded that Ad5 neutralizing antibodies to fiber are more common than to hexon in the naturally infected population, but that immunization with a replication-defective Ad5-based vector raised more neutralizing antibodies to capsid proteins other than fiber.¹⁰⁹ Most studies in animal models infected with Ad5-based vectors have found that the predominant neutralizing antibodies are directed at hypervariable regions in hexon.⁴⁰⁴,⁵⁹¹,⁸¹⁰ Mechanisms by which neutralizing antibodies function include virus aggregation, virus destabilization, and blocking virus receptor interactions and integrin-mediated internalization (reviewed in⁶⁷⁰). One study with the Ad5 neutralizing hexon monoclonal antibody 9C12 reported that following infection and endosome penetration of the virus–9C12 complex, microtubule-dependent translocation to the microtubule-organizing center was inhibited.⁶⁷⁰ Another study with the same monoclonal antibody as well as polyclonal Ad5 neutralizing antibodies concluded that the cellular cytosolic protein TRIM21 binds to the antibody in the internalized antibody–virus complex and targets the virus to the proteasome for degradation.⁴⁵⁴

There are group- and type-specific epitopes on both hexon and fiber. Type-specific domains have been mapped to unique sequences in loop 1 (amino acids 281 to 292) and loop 2 (amino acids 441 to 455) of hexon by generating neutralizing antibodies to peptides from each of these regions. This epitope is referred to as the Ε determinant.⁴⁴⁸ Loops 1 and 2 had previously been shown to be on the surface of the virion by crystallography. Differences in the HA properties of rhesus and vervet erythrocytes for two important subtypes of Ad11 (Ad11p and Ad11a) have been related to nucleotide sequence differences in the shaft and knob region of fiber. The knob region of fiber, which has HA properties that are used for HI (hemagglutination inhibition) tests, includes the γ determinant.⁴⁴⁸ Ad11p and Ad11a have some differences in tissue tropism in that Ad11p can be persistent in the urinary tract and Ad11a causes acute respiratory tract infections. These properties of the whole virus may be related to the changes in fiber polypeptides that are otherwise identical for 92.3% of their amino acids.⁴⁶⁸

There are group-reactive antibodies that react with conserved domains of hexon from all human serotypes.⁵²⁹ These interactions were classically measured by the complement fixation (CF) test and were useful in identifying an agent as an Ad. Subsequent serologic techniques, such as immunofluorescent (IF) antibody and enzyme-linked immunosorbent assay (ELISA) determinations, were also capable of detecting group reactivity shared by most of the human Ads.

Ad-specific CD4+ T lymphocytes have been detected in peripheral blood monocytes (PBMCs) in nearly all naturally infected humans of all ages (reviewed by⁴⁰⁹).⁸⁴,¹⁰²,²⁷⁸,³²¹,⁴¹⁴,⁵³⁶,⁵⁴¹,⁵⁴²,⁷⁵⁰ When PBMCs were stimulated in bulk culture (e.g., by incubation with intact Ad particles, Ad-infected cell extracts, or purified Ad proteins or with pools of peptides that span various Ad proteins), CD4+ T lymphocytes specific to hexon were identified from healthy donors.¹⁹⁹,⁵⁴⁰,⁵⁴²,⁶³⁶,⁷⁵⁰,⁸¹⁴ Other studies using peptides corresponding to parts of several Ad proteins identified a number of CD4+ T-cell epitopes, including the dominant human leukocyte antigen (HLA) DP4-restricted H910-924 epitope, located in the base of the hexon protein, conserved among Ad serotypes, and detectable in PBMCs from 78% of healthy adults analyzed.⁵⁴⁰,⁷⁰⁰ Many other CD4+ T-cell epitopes located in the conserved regions of Ad5 hexon and that are conserved across Ad serotypes have been identified that are recognized by PBMCs.⁴¹⁰,⁴¹¹,⁶³⁶,⁷⁵⁰,⁸¹³ In one study in which PBMCs from 44 healthy donors were analyzed, 10 CD4+ T-cell immunodominant hexon epitopes were detected in more than 50% of subjects examined.⁶³⁶ The HLA restriction element for some of these peptides is known.⁶³⁶,⁸¹³ Although not as frequently found as are Ad- and hexon-specific CD4+ T cells, hexon-specific CD8+ T cells have also been detected in PBMCs from healthy donors; the cells are cross-reactive against various Ad serotypes, they secrete interferon-γ, and they have cytolytic activity in culture.¹⁹⁹,³²⁰,³²¹,³⁴⁵,⁴¹⁰,⁴¹⁴,⁶⁶⁹,⁷⁰⁰,⁸¹³ CD4+ and CD8+ T cells specific to Ad proteins other than hexon, including penton base⁴¹¹ and the DNA polymerase,³⁴⁴,³⁴⁵ have also been identified.¹⁹⁹ There is increasing evidence that cytotoxic T lymphocytes (CTLs) specific to hexon are protective in humans.⁴¹⁰ Indeed, T-cell “lines” (mixture of CD4+ and CD8+ cells) specific to hexon (and other Ad proteins) have been shown to be effective by adoptive cell transfer in treating infection by various Ad serotypes in allogeneic hematopoietic stem cell transplant patients.¹⁹⁸,⁴¹¹,⁴¹³,⁸¹³ The therapeutic effect is believed to be through the coordinated action of the adoptively transferred CD4+ and CD8+ T cells.⁸¹³

Human Ads are mostly species specific in their replication cycle, as are most Ads of other mammals or avian species. Even nonhuman primates are poor hosts for human Ads. Human species C Ads can replicate in the lung of cotton rats,²²⁷ and the pathogenesis of Ad pneumonia has been studied in these animals.⁵⁴,²²⁷,⁵⁷⁴ An ocular model of infection with either Ad5 or Ad8 has been described in cotton rats, and the clinical manifestations, including subepithelial corneal opacities, were similar to epidemic EKC.³⁵⁹,⁷³⁰ The animals shed virus, developed specific antibodies to the infecting virus, and were able to spread the infection to control cotton rats. A similar ocular model was reported in New Zealand White rabbits after topical or intrastromal inoculations of Ad5.⁶⁰²,⁷²⁹ The virus appeared to replicate, and most of the animals developed a humoral immune
response. There were findings of blepharitis, conjunctivitis, iritis, corneal edema, and subepithelial corneal infiltrates that were consistent with immune-mediated clinical disease. The Ad5 New Zealand White rabbit model has been used to evaluate the anti-Ad activity of several compounds⁶⁰¹ including cidofovir and 2′-3′ dideoxycytidine,⁶⁰³ N-chlorotaurine,⁶⁰⁴ and dexamethasone povidone-iodine.¹²³ Ad5 can also replicate in porcine tissues.⁷²³

A number of groups have explored the mouse as a model for pathogenesis of human Ads or as a model to evaluate oncolytic (replication-competent) Ad vectors for cancer gene therapy. The results from these studies have been mixed. Human Ads can infect cells of virtually all mammalian species including the mouse, especially if high multiplicities of infection are used (∼100 plaque forming units [pfu] per cell). The early proteins (synthesized prior to Ad DNA replication) are expressed at good levels, but most workers have found that Ad DNA does not replicate (or barely replicates), and therefore that late genes are not expressed (or are expressed at very low levels). Nevertheless, there are reports that Ad5 or Ad2 can replicate to low levels in several mouse carcinoma cell lines²²¹,²⁵⁸,⁵⁰³,⁷⁷²; however, this is not a universal finding (e.g.,³⁴¹). When high doses of Ad5 were inoculated intravenously into CBA mice, there appeared to be several orders of magnitude of replication in the liver.¹⁶⁹ On the other hand, no replication was seen in the lungs of C57BL/6 mice following intranasal administration of Ad5.²²⁸ Also, little or no replication was observed in any organs following intravenous administration of an Ad5-based oncolytic vector in C57BL/6 mice⁸⁰¹ (this vector retains all Ad genes except some in the E3 region).

Ad5 replicates modestly in cotton rat cells,⁷²⁶,⁶⁸² canine cells,⁷⁰³ porcine cells,³⁴¹ and Syrian hamster cells.⁷⁰⁵ In three Syrian hamster cancer cell lines, the burst size (virus yield per cell) was about 1,000, only 10-fold less than in A549 cells.⁷⁰⁵ In Syrian hamsters, about four orders of magnitude of Ad5 replication were seen in the lung following intranasal or intratracheal administration.²⁹²,⁴⁸⁷,⁷⁰⁵ Ad5 also replicates in the liver of Syrian hamsters following intravenous administration.⁸⁰¹

Because Syrian hamster tissues are quite permissive for Ad5 and there are numerous Syrian hamster cancer cell lines, Syrian hamsters have been used as a model to investigate the toxicology and antitumor efficacy of oncolytic Ad5-based vectors.¹⁵⁷,³⁶⁹,⁷⁰⁵,⁷⁰⁷ Immunocompetent and immunosuppressed (by treatment with cyclophosphamide) hamsters bearing subcutaneous tumors formed by injection with various Syrian hamster cancer cell lines were treated by intratumoral injection with a variety of oncolytic Ad vectors.⁶³,⁶⁴,⁶⁶,⁹⁴,⁹⁵,¹⁵⁵,¹⁵⁶,³⁶¹,³⁸⁴,⁶⁴⁸,⁶⁷⁷,⁷⁰⁵,⁷⁰⁶ In general, these studies show that oncolytic Ad5-based vectors suppress the growth of tumors, and that there is a rapid adaptive immune response to the vector that appears to eliminate the vector from the tumor. Syrian hamsters have also been used to study the biodistribution and toxicity of oncolytic Ad5-based vectors in advance of clinical trials.³⁷⁴,⁴²⁹,⁴⁶⁰,⁶⁷⁵,⁸⁰¹

Immunocompetent,¹⁵⁹,⁷²⁷ newborn,⁸¹⁵ and immunosuppressed⁷²⁷ Syrian hamsters have been used to evaluate compounds to inhibit Ad5 replication. A mouse model was used similarly to test cidofovir against disseminated mouse Ad type 1 infection.⁴¹⁷

Human Ads inoculated into a variety of rodent species cause tumors. Ad12, 18, and 31 are highly oncogenic in newborn Syrian hamsters, and much has been learned about the mechanism of action of the genes in the E1A and E1B regions from these viral models (see Chapter 55). The integrated Ad12 sequences in these hamster tumors are a model to understand epigenetic consequences of foreign DNA integrations.¹⁶⁰ Ad9 causes fibroadenomas and mammary sarcomas in rats, and an early region 4 gene seems to be important in this model.⁴⁰⁵

The proteins coded by the Ad E3 transcription unit are believed to provide protection of infected cells from the host antiviral response (Fig. 56.3) (reviewed in⁵⁷,¹⁹⁶,²⁵⁹,³⁰⁹,⁴³⁰,⁴⁴⁵,⁴⁶⁵,⁶⁴⁶,⁷⁸⁵). The Ad2 or Ad5 E3-gp19K protein is a type I glycoprotein localized in the endoplasmic reticulum (ER) (reviewed in²⁵⁹). E3-gp19K binds to major histocompatibility complex class I (MHC-I) heavy chain in the ER, prevents transport of MHC-I to the cell surface by virtue of an ER retrieval signal on E3-gp19K, and prevents killing of Ad-infected cells by CTLs.¹⁴,⁸²,¹³¹,⁵⁸² E3-gp19K and its function are conserved among serotypes in all human Ad species²¹² except species A in which the E1A proteins cause down-regulation of MHC-I.³⁴⁰ E3-gp19K binds with higher affinity to HLA-A than to HLA-B, and it binds poorly if at all to HLA-C.²¹²,²⁵⁹ E3-gp19K binds via a conserved domain (among Ad serotypes)²¹³,⁶⁴¹ to the outer surface of the peptide-binding groove on MHC-I molecules.²⁵⁹,⁴³⁷ E3-gp19K also binds to transporter associated with processing (TAP), prevents formation of the TAP–tapasin complex, and limits the inclusion of TAP into the peptide-loading complex of antigenic peptide, MHC-I, and chaperones.⁵² This property of E3-gp19K could possibly retard cell surface expression of MHC-I in individuals with HLA-B and HLA-C MHC-I molecules.²⁵⁹

Reduced expression of MHC-I could render Ad-infected cells susceptible to killing by natural killer (NK) cells. To potentially enhance this possibility, the Ad E1A proteins up-regulate ligands recognized by the NKG2D receptor on NK cells and sensitize the E1A-expressing cells to NK cell–mediated cell lysis.⁴⁶⁷,⁶¹¹ Multiple ligands for NKG2D are known, including the MHC-I chain-related A (MICA) and B (MICB) proteins. As is the case with MHC-I, E3-gp19K causes retention of MICA and MICB in the ER, prevents their transport to the cell surface, and reduces killing of E3-gp19K–expressing cells by NK cells.⁴⁶⁷,⁶⁴¹

The Ad E3-14.7K protein inhibits tumor necrosis factor (TNF)-induced cytolysis of Ad-infected cultured cells.²³²,²³²,²³⁴,³⁰⁵,³⁰⁶,⁵⁷⁹ E3-14.7K binds to cellular IKKγ/NEMO³⁷³,⁴²⁶ and is reported to modulate NF-kB activity and inhibit TNF-induced apoptosis.⁴²⁶ E3-14.7K also is reported to bind to caspase 8.¹⁰⁷,³⁷³ In addition, E3-14.7K inhibits internalization of TNF receptor 1 as well as formation of the death-signaling complex (DISC) that is required for TNF-induced apoptosis.⁶²⁵ Two studies report that E3-14.7K inhibits TNF-induced signaling through NF-kB,⁹⁰,⁴²⁶ whereas another study did not observe such activity for E3-14.7K.⁶²⁵ In a recent report, E3-14.7K stably expressed in mouse cells inhibited induction by TNF of the chemokine CCL2 (monocyte chemoattractant protein-1 [MCP-1]) by preventing phosphorylation of glycogen synthase kinase-3β and recruitment of NF-kB to the CCL2 promoter.⁶⁷⁸ Some researchers¹⁰⁷,⁷⁹⁴ but not others⁷¹⁴ report that E3-14.7K inhibits apoptosis by Fas ligand. E3-14.7K also inhibits TNF-induced release of arachidonic acid,³⁹⁰,⁷⁰⁹,⁸²² a property that might be important in reducing inflammation.

Two other E3 proteins, of 10,400 daltons (10.4K) and 14,500 daltons (14.5K), which were originally named E3-10.4K⁷¹⁶ and E3-14.5K,⁷¹⁵ respectively, form a molecular complex originally named E3-10.4/14.5K.⁷¹⁹ The E3-10.4K protein⁸⁸ and then later the E3-10.4K/14.5K complex⁷¹⁹ were reported to be required to clear the epidermal growth factor receptor (EGFR) from the cell surface (Fig. 56.4). The E3-10.4K and E3-14.5K proteins were later renamed receptor internalization and degradation (RID)α and RIDβ, respectively, when it was shown that these proteins also are able to cause the internalization and lysosomal degradation of not only EGFR but also cell surface Fas,¹⁸⁰,⁴⁶⁴,⁶⁶²,⁷¹⁴ TRAIL receptor 1,⁴⁹,⁷²⁰ and TNF receptor 1.¹¹⁰,¹¹¹ RID inhibits cytolysis induced by Fas ligand (i.e., by an agonist antibody)⁴²⁸,⁶⁶²,⁷¹⁴ and TNF.²³³,³⁹⁰,⁸¹² RID, functioning in concert with the E3-6.7K (6,700 daltons) protein, also down-regulates TRAIL receptor 2 from the cell surface.⁴⁹,⁴²⁷ One laboratory⁴⁹ but not another⁴²⁷ reported that E3-6.7K is required together with RID to down-regulate TRAIL receptor 1. A cytosolic tyrosine sorting motif (YXXφ) in RIDβ¹¹⁰,⁴²⁸ and a cytosolic dileucine motif ²⁸⁶ and tyrosine-containing motifs¹¹⁸ in RIDα are required for these proteins to function.

RID also has been shown to inhibit TNF-induced translocation of cytosolic phospholipase A₂ to membranes (from where arachidonic acid is generated)³⁹⁰ and to inhibit
lipopolysaccharide- and interleukin-1β–mediated signaling responses.¹⁵⁰ Thus, both RID and E3-14.7K might function in vivo to inhibit inflammatory responses associated with infection.

In contrast to most laboratories who have studied RID, one laboratory has reported that RIDα can function in the absence of RIDβ to down-regulate EGFR.¹¹⁸,²⁹³,⁶⁴⁵ This laboratory also reported that RIDα alone is able to regulate endosome maturation by mimicking guanosine triphosphate (GTP)-Rab7⁶⁴⁵ and to activate an autonomous cholesterol regulatory mechanism.¹¹⁷ Possible explanations for how RIDα, RIDβ, and the RIDα/RIDβ complex (i.e., RID) could function independently have been discussed.⁶⁴⁵

As mentioned, the E3-6.7K protein is required together with RID to down-regulate TRAIL receptor 2 from the cell surface in Ad-infected cultured cells.⁴⁹,⁴²⁷ In transiently or stably transfected Jurkat cells, E3-6.7K alone inhibited apoptosis induced through Fas, TNF receptor, and TRAIL receptors and prevented TNF-induced release of arachidonic acid.⁴⁸⁰ Further, E3-6.7K was shown to interact with calcium modulator and cyclophilin ligand (CAML), a calcium-modulating protein,²⁴¹ to prevent calcium efflux from the ER, maintain calcium homeostasis, and inhibit apoptosis induced by thapsigargin (which inhibits calcium uptake by the ER).²⁴¹,⁴⁸⁰

The functions for the E3 proteins as described previously have been determined by studies in cell culture, and the question arises as to whether the E3 proteins have these same functions or other unknown functions in vivo (animal models and humans). Some and perhaps all of the E3 proteins are not essential for Ad5-based oncolytic vectors to replicate in Syrian hamsters⁷⁰⁵ or in cancer patients, but there is good evidence that E3 proteins have functions in vivo. In one study, expression of all the E3 proteins from a replication-defective Ad vector prolonged transplants of human cells in immunocompetent mice.⁷²⁴ In another study, the entire Ad2 E3 region stably expressed in an immortalized Gunn rat hepatocyte cell line protected against allograft rejection following transplantation into rats.⁴⁵⁸ This protection correlated with reduced Fas expression and inhibition of Fas-mediated apoptosis in the transplanted cells (which could be mediated by RID). Expression of E3-gp19K plus RID by lentivirus vectors in an insulin-producing β-cell line corrected diabetes in allogeneic mice, whereas the cell line without E3-gp19K plus RID did not protect.³⁸³ In transgenic mice, the entire Ad2 E3 region that was placed under the control of the rat insulin promoter (RIP) for targeted expression in murine pancreatic islet cells prevented autoimmune diabetes in a virus-induced (lymphocytic choriomeningitis virus) murine model of type I diabetes or in nonobese diabetic (NOD) mice.¹⁷⁹,⁷⁵⁵ In further studies in which all E3 genes except E3-gp19K or except the E3B region genes (E3-RID plus E3-14.7K) were expressed from the RIP in transgenic mice, one study found that E3-gp19K but not the E3B region genes prevented autoimmune diabetes,³¹⁰ but another study obtained the opposite result.⁵⁶⁶ In studies addressing E3-14.7K, the gene for E3-14.7K, which was cloned into vaccinia virus together with the tnf gene, increased the virulence of the vaccinia virus carrying the tnf gene alone by reversing the antiviral effects of TNF.⁷³¹ In other studies, a transgenic mouse was constructed in which E3-14.7K is expressed selectively in the distal respiratory epithelium (alveolar and bronchial) from the surfactant protein B promoter.²⁶⁴ E3-14.7K protein suppressed pulmonary epithelial cytotoxicity and lung inflammation in response to respiratory infection with a replication-defective Ad vector or to intratracheal administration of lipopolysaccharide.²⁶⁴ In an influenza virus study, E3-14.7K reduced expression in the lung of CCL2, a chemokine that functions in recruitment of inflammatory monocytes and lymphocytes.⁶⁷⁸ E3 genes were required to reduce inflammation in an Ad5 pneumonia model in cotton rats²²⁷ and mice.⁶⁷⁶ The E3-6.7K plus E3-gp19K proteins were required to prolong persistence of an oncolytic Ad in Syrian hamsters.⁶⁵ These various studies suggest that E3 proteins function in vivo in a manner consistent with their functions seen in vitro.

Ads enter susceptible hosts by the mouth, nasopharynx, or ocular conjunctiva. Recent experiments have defined the Ad receptors (Fig. 56.5) (reviewed in⁴⁰⁶,⁴⁶⁹,⁵²¹,⁷⁷⁰,⁸¹⁷). The cell protein named CAR (coxsackie-adenovirus receptor) is a receptor shared by these two groups of unrelated viruses and is the target of fiber in many if not all serotypes in species A, C, D, E, and F.⁵⁵,⁵⁵,⁵⁹⁷ CAR is present on tight junctions of polarized cells where it mediates cell–cell adhesion. Species B Ads can be differentiated based on their receptor usage.⁷³² Species B, group 1 (Ad16, 21, 35, 50) viruses nearly exclusively utilize CD46 as a receptor.²⁵³,⁴⁵⁷ Initially, Ad37, a species D Ad, was also thought to use CD46,⁷⁹¹ but recent data suggest that GD1a glycan is the more likely major receptor for this virus.⁵²⁵ CD46 (also known as membrane co-factor protein) is expressed in virtually all cells where it acts as a co-factor for inactivation of the complement components C3b and C46. Species B, group 2 (Ad3, Ad7, 14) viruses share desmoglein 2 as the high-affinity receptor.⁷⁶⁹,⁷⁷⁰,⁷⁷¹ Desmoglein 2 is a calcium-binding transmembrane glycoprotein belonging to the cadherin protein family. When the Ad fiber binds to desmoglein 2, there is opening of intercellular junctions that results in increased access to receptors trapped deep within the junction (i.e., CD46 and Her2/neu).⁷⁷⁰ Further, such dissociation of the intercellular junctions may facilitate the lateral viral spread in epithelial cells and, potentially, the penetration of Ad into subepithelial cell layers and the bloodstream. Lastly, species B, group 3 (Ad11) viruses preferentially interact with CD46 but also utilize desmoglein 2 if CD46 is blocked.⁷⁷⁰

Heparin sulfate glycosaminoglycans, which are long heterogeneous, heavily sulfated carbohydrates that are abundant within the extracellular matrix, can mediate the CAR-independent binding of Ad2 and Ad5 to cells, but it is not known if these are bona fide receptors.⁸¹⁷ The class I MHC molecule has been proposed as a second receptor for Ad5³⁰⁴ but has not been confirmed by other groups.¹⁴²,⁴⁶³ Three species D Ads, Ad8, Ad19, and Ad37, all major causes of EKC, appear to bind to α2,3-linked sialic acid present in the GD1a ganglioside on the corneal cell surface.⁵²⁵ Several integrins participate in Ad uptake into cells by interacting with the RGD motif on the Ad penton base protein.⁸¹⁷

Most of the studies described previously were conducted in vitro. Recent studies of systemic administration of Ad vectors in mice raise the possibility that infection of tissues in vivo may be different (see section on Adenoviruses as Vectors for Vaccination and Gene Therapy). These studies propose a CAR-independent model in which Ad infection of murine hepatic cells occurs through binding of Ad to blood factors, especially factor X, directing the complexes to hepatocellular receptors including heparin sulfate proteoglycans (Fig. 56.5). Whether this pathway operates in humans is a key²⁸¹ question. Recent studies clearly demonstrate that all of the tested species of Ads, except for the species D viruses, efficiently bind human factor X.⁷⁵⁷ The high-affinity interaction of Ads with factor X may facilitate “bridging” the hexon protein in the Ad capsid to heparin sulfate proteoglycans expressed on the surface of hepatocytes.³⁵²,⁷⁵⁷ The Ad–factor X complex binds to the cell surface through the serine protease domain of factor X and not through a direct interaction of the virus with the cell surface.⁷⁵⁷ Once bound to the cell surface, efficient and rapid intracellular transport of the Ad, though, remains dependent upon engagement of α_v integrins via the penton base protein.⁷¹

The lower serotypes, Ad1, 2, 5, and 6, are ubiquitous, particularly in young children, and may be shed for months, especially in the stool, which is probably responsible for the endemic spread of these agents by the fecal–oral route to new pools of susceptible infants and children.²⁰⁵ An epidemic form of Ad keratoconjunctivitis (EKC) has been spread in swimming pools²⁰⁷ from contaminated water and in medical settings³³⁶ from Ad-infected ophthalmologic instruments. A third pattern of spread was unique in the military setting, occurring during the early period after induction into service. Ad4 and Ad7 caused acute respiratory distress (ARD), including pneumonia, and were the result of air-borne inoculation of the respiratory tract¹⁶⁸ as well as acquisition after contact with contaminated surfaces in living quarters.⁶¹⁵

It is clear from the original findings of Ads in tonsils and adenoids that these tissues of the oropharynx are a major initial site of replication for the entering Ads. For serotypes causing
respiratory disease, the initial replication most likely occurs in the nonciliated respiratory epithelium, although some limited replication and persistence can also occur within lymphocytes (reviewed in⁴⁴⁵). These conclusions are based not on careful observations of cells in situ but on the cells in which Ad replicates in tissue culture. Ciliated respiratory epithelium of the lower airway is difficult to infect with Ads through the apical surfaces, which do not contain the CAR receptor; however, disruption of the integrity of cell–cell contact allows basolateral infection of such polarized epithelial cells using CAR.⁵⁶⁵,⁷⁶⁸

Most of the manifestations of Ad infection are locally in the eyes and pharynx, but contiguous extension into the lungs results in some cases. One possible molecular mechanism facilitating the spread of the virus over respiratory epithelia is that the fiber protein, which is synthesized in great excess in the infected cell and is released when the cell is lysed, binds CAR on the basolateral surface. This binding disrupts the CAR homodimers in tight junctions, thus increasing paracellular permeability. This in turn allows the virus to escape onto the apical surface of the respiratory epithelium, thereby making it possible for the virus to infect other areas of the respiratory tract.⁷⁶⁷ Likewise, for the species B viruses that bind to desmoglein 2, binding to desmoglein 2 resulting in dissociation of the intercellular junctions may facilitate spread of the virus locally and into deeper tissue layers, allowing access to the bloodstream.⁷⁷⁰ Even in the nonreplicating mouse model of human Ad infection, the virus can cause inflammation in the lungs, which is strong evidence for direct extension into these target organs.²²⁸,⁶⁷⁶ Ads have been cultured from the blood during fatal Ad respiratory disease, suggesting viremic spread in some situations (see the section later on immunocompromised patients). As CAR is present on endothelial cells, viremia might be promoted by the fiber protein through the mechanism described earlier. The successful use of oral, live, microencapsulated Ad vaccines by the military to prevent ARD suggests that if the respiratory tract can be physically bypassed by Ad4 and Ad7, intestinal replication of the virus causes an immunizing rather than a virulent infection.¹²⁹ Although most Ads replicate in the intestine without causing gastroenteritis, Ad40 and 41 are responsible for intestinal disease. Ad disease in the urinary bladder, primarily by species B Ads in immunocompetent hosts, suggests that the virus probably is viremic at some stage in order to reach this organ. There is no evidence for ascending infection for these species B serotypes, which are less commonly found in the intestine and are more common in young males than in females. The route of infection of the liver, especially in immunosuppressed liver transplant recipients, is unknown, but some patients might be infected by latent Ads that are present in cells such as lymphocytes in the transplant.

Various aspects of the immune response to Ads have been described in other sections, as have mechanisms that the virus uses to evade the immune response (see Fig. 56.3 and section on Functions of Adenovirus E3 Proteins). The innate response to Ad also has been reviewed⁴³⁹,⁵⁰⁴ (see section on Adenoviruses as Vectors for Vaccination and Gene Therapy). In the airways, the virus must penetrate the surface fluid, and sialic acid present in the mucus may bind and inhibit species D Ads that use sialic acid as a receptor.³⁴²,⁵²⁵ The virus must also survive chemical defenses of the host. These include a large variety of antimicrobial peptides that are able to neutralize microbes directly. Among these peptides are the defensins, a family of small cationic amphipathic peptides divided into two classes, α-defensins and β-defensins (reviewed in²⁷³). The α-defensins HNP1 and HD5 were shown to neutralize Ad serotypes in species A, B1, B2, C, and E, but not in species D and F.²⁷⁰,⁶⁷¹,⁶⁷² The defensins bind Ad particles outside the cell, block uncoating of the virion, and restrict the release of virions from endocytic vesicles.⁶⁷¹ Thousands of molecules of α-defensins bind to a sensitive serotype; neutralization of the virus depends on binding to critical determinants in a region spanning the fiber and penton base proteins.⁶⁷² Binding to these determinants is proposed to prevent the release of fiber from the virion, the first step in the virion uncoating process within the endosome.⁶⁷² HNP1 is expressed primarily in neutrophils, monocytes, lymphocytes, and natural killer cells, while HD5 is expressed mainly by Paneth cells in the intestine.²⁷³,⁶⁷¹ It is not known if these or other defensins play a role in Ad infections in humans.

With respect to cellular defenses, alveolar macrophages and Kupffer cells play an important role in elimination of Ad vectors from the lung and liver, respectively, in murine models⁴³⁹,⁵⁰⁴ (Fig. 56.6) (see section on Adenoviruses as Vectors for Vaccination and Gene Therapy). These cells take up the vectors rapidly and secrete inflammatory cytokines such as TNF, interleukin-6 (IL-6), and IL-8. A robust inflammatory response characterized by early cytokine (IL-1β, IL-6, interferon-γ [IFN-γ], IL-12, and TNF) and chemokine release as well as neutrophilic and monocytic infiltration have been described in the murine pneumonia model after nonpermissive infections with species C Ads.²²⁸,³⁴⁹ Increased levels of IL-6, IL-8, and TNF have also been associated with Ad infection in children.⁴⁷⁶ A recent study suggested that there may be specific cytokine signatures in pediatric stem cell transplant recipients with localized and invasive Ad infection. Patients with invasive infections had increased levels of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, IFN-γ, and TNF-α, as well as IL-17, macrophage inflammatory protein-1α (MIP1α), oncostatin M (OSM), and IFN-γ–inducible protein 10 (IP10).²⁷² Invasive Ad infection was also associated with the simultaneous release of the cytokines IL-1β, IL-17, IL-18, OSM, MIP-1α, and IP10.²⁷²

Ad infection of cultured cancer cells, respiratory epithelial cells, hepatocytes, and antigen-presenting cells activates the NF-kB and MAP kinase pathways.⁵⁴⁷ Induction of IL-6 and IL-8 does not require Ad gene expression and likely occurs via interaction of the virion with integrins on the cell surface. A biphasic mononuclear cell response in the cotton rat suggested an early appearance of monocytes and NK cells followed by specific T and B lymphocytes. The cell infiltrate was changed from a mononuclear to a polymorphonuclear leukocyte response by deletion of the Ad E3 anti-TNF genes (14.7k, ridα, ridβ). Deletion of the anti-MHC Ad E3-gp19K protein increased the intensity of the inflammatory response in the cotton rat but not in the mouse.²²⁷,⁶⁷⁶ These observations suggest that immunopathology plays a role in the host response to
Ad infection, and this process is ameliorated by the E3 inhibitors of the host inflammatory response.²²⁷,⁵⁷⁴ Another Ad protein, the L4-100K protein, which is expressed at late stages of infection, inhibits granzyme B–mediated death by CTL.¹⁶ Thus, it is not clear at this point whether the cell-damaging effects of Ad infection or the host immune responses to the pathogen are responsible for the tissue pathology and clinical manifestations.

Induction of type I interferons (α and β) is part of the innate response to Ad infection. Ads have at least two mechanisms to counteract these interferons (see Chapter 55). First, the E1A proteins block the assembly of interferon-induced transcription factors, and second, VA-RNA_I, a 159-nucleotide RNA, binds to the protein kinase named PKR, prevents its activation by double-stranded RNA, and thereby prevents PKR from phosphorylating eukaryotic initiation factor eIF2 and shutting down protein synthesis.

Neutralizing and nonneutralizing antibodies are discussed in the section, Description of Key Antigens. After infection, most patients develop both group- and type-specific antibodies to the infecting Ad strain. Group-specific antibodies are not neutralizing of viral infectivity but are useful in measuring patient response when infection with an Ad is suspected but no isolate was obtained. In this case, acute and convalescent sera taken several weeks apart may demonstrate rising antibodies to a group-specific Ad antibody such as that measured by CF, IF, or ELISA, using a viral-infected tissue culture extract or purified hexon from a single Ad type. Type-specific antibodies are measured by either the neutralization test or inhibition of HA, as described in the section Viral Serology. It is clear that patients may continue to shed Ads intermittently, especially in their stools, for many months after a successful humoral immune response has occurred.²⁰⁵ Neutralizing antibodies may be protective against disease manifestation in the previously infected host or against reinfection with the same serotype, but they do not eliminate the carrier state.

Recent studies have begun to define the T-cell response to Ad infection in humans and Ad (see the section on Description of Key Antigens) (reviewed in³⁸²,⁴⁰⁹,⁷⁶²). Although most of the early work in humans described Ad-specific CD4+ T cells, recent studies have detected and characterized Ad-specific CD8+ T cells. These CD4+ and CD8+ T cells are frequently cross-reactive among Ad serotypes, and many of the epitopes have been mapped to the conserved regions of hexon.

Although it is not known if these T-cell epitopes are protective against Ad in humans, most likely T cells in general are protective because the absolute lymphocyte level and the CD4+ T cell level correlate inversely with Ad infection and disseminated adenoviremia in immunosuppressed transplant patients (see section on Diseases Associated with Immunocompromised Patients). For example, in adult stem cell transplant recipients, clearance of human Ad viremia coincides with emergence of a coordinated CD8+ and CD4+ T-cell response against Ad hexon epitopes.¹⁹⁹,²⁷⁷,⁸¹³

Although donor lymphocyte infusions can be used in hematopoietic stem cell transplant recipients with Ad infections to improve infectious diseases outcomes, potentially life-threatening graft-versus-host disease (GVHD) may develop.¹²⁵ Adoptive transfer of CTL against Ad can rapidly reconstitute anti-Ad immunity after stem cell transplantation without causing GVHD.¹⁹⁹,²⁷⁷,⁴¹⁰,⁸¹³ Methods are being developed to activate and expand T cells in vitro, and these interventions appear to be a promising potential therapy for immunocompromised patients with serious Ad infections (reviewed in⁵,³⁶⁷,⁴⁰⁹,⁴¹²,⁴⁷⁴,⁸¹³).

It is not known why certain serotypes characteristically cause disease in some organs and not in others. Tissue culture experiments and the existing animal models do not explain the mechanisms of such tissue tropism or organ-specific pathogenicity. There is nothing obvious about the bioinformatics analysis of multiple Ad genomes that explains differences in pathogenicity, although most differences among species are in the E3 region.¹⁴¹ Because most Ads can attach to the CAR receptor, it is unlikely that this receptor can explain differences in the in vivo tropism of the various serotypes. Even the oral vaccine strains used to prevent respiratory disease in military personnel seem to depend more on physical formulation (i.e., enteric coating of a live virus so that it bypasses the oropharynx and first exposes the intestinal epithelium) than on mutation of virulence genes.⁴⁴⁶

Acute hemorrhagic cystitis, an illness occurring almost exclusively in boys and associated with Ad11, is characterized by gross hematuria.⁵³⁰ Its significance lies in the potential confusion with other, more serious diseases of the kidney (such as glomerulonephritis). This self-limited disease is usually not accompanied by fever or hypertension, and tests of renal excretory and concentrating functions have been essentially normal. Ad21, like Ad11, is a species B HA type I and can also cause hemorrhagic cystitis.⁴⁹³ In Japan, when acute hemorrhagic cystitis occurs in a boy between the ages of 6 and 15 years, an Ad isolation from urine or a rise in neutralizing antibody occurs in about 70% of patients.⁵³⁰ In comparable studies of hemorrhagic cystitis in the United States, only 20% of cases can be linked to an acute Ad infection, and for 60% of the total, the etiology remains unexplained.⁴⁹³

The other population that develops hemorrhagic cystitis is immunosuppressed transplant recipients. According to one study, Ad infections account for 3.9% of hemorrhagic cystitis among pediatric hematopoietic stem cell transplant (HSCT) recipients.²³⁵ Another study of mostly adult HSCT recipients found that Ad was associated with 9.8% of cases of hemorrhagic cystitis.⁴⁸⁶ Ad hemorrhagic cystitis in HSCT patients is more frequently associated with T-cell purging and was less common in patients with acute GVHD than other causes of hemorrhagic cystitis.⁴⁸⁶ In the kidney transplant population, Ad can also cause hemorrhagic cystitis with or without concomitant nephritis; such patients typically present with fever and often feel poorly.¹²,²⁶¹,²⁸²,³⁸⁰,⁵⁴⁴ In some patients, it appears that the infection was introduced with the transplanted kidney.³⁸⁹,⁷⁴⁶ In addition to Ad11, two other species B serotypes, Ad34 and Ad35, were isolated first from renal transplant recipients. Ad34 was isolated from urine, whereas type 35 was isolated from kidney and lung tissue at autopsy.²⁸³,⁵⁰⁰ Although neither was accompanied by the symptoms of hemorrhagic cystitis, the Ad35 clearly contributed to the patient’s demise from pneumonia. Ad34, 2, and 31 were isolated from allogeneic bone marrow, hematopoietic stem cell, and liver transplant recipients with hemorrhagic cystitis, respectively.⁴⁸⁵,⁶³²

It is rare to isolate any of the Ads from either the cerebrospinal fluid (CSF) or the brain. However, several reports have directly demonstrated Ads in CSF (Ad3, 5, 6, 7, 7A, 12, and 26).¹¹³,¹⁶⁶,³⁶⁴ One patient with malignant lymphoma, immunosuppressed by chemotherapy, had an Ad32 isolated from the brain at autopsy.⁶⁰⁶ A patient with large B-cell lymphoma developed meningoencephalitis due to Ad7.²⁰⁰ Ad5 was cultivated from CSF in two immunocompetent patients, one with meningoencephalitis and the other with meningitis.⁶⁷⁴ A bone marrow transplant developed fatal subacute Ad meningoencephalitis.¹⁴⁰ A 12-year-old immunocompetent girl developed tubulointerstitial nephritis with acute renal failure, hepatitis, and meningoencephalitis following systemic Ad infection; she recovered with supportive care.¹⁸⁶ There are other cases of meningoencephalitis in which viral isolation from extraneural sites or antibody titer increases have been used to make a diagnosis, especially associated with epidemic Ad7 pneumonia in children.⁶⁶⁴ A case of sudden unilateral deafness was associated with an Ad3 infection of the nasopharynx.³³⁰

Gastroenteritis, or inflammation of the stomach and small and large intestines, is characterized by fever, vomiting, and diarrhea. It is frequently caused by viruses, bacteria, or parasites. Viruses that cause gastroenteritis include rotaviruses, Ads, noroviruses, calciviruses, astroviruses, and Norwalk virus. Rotaviruses are the leading cause of diarrhea in the world, and calciviruses cause the most gastroenteritis outbreaks in industrial nations.²²⁹ The role of Ads is discussed in the next few sections.

Myocarditis is an inflammatory disorder of the myocardium characterized by necrosis of myocytes and infiltration of inflammatory cells. The most common form of this disease, viral myocarditis, is generally thought to be caused by enterovirus infection (e.g., coxsackievirus), although there is evidence that Ad may be a significant cause as well (reviewed in⁶⁹,³⁵⁶,⁴⁵³). Ads do not normally cause symptomatic infection of the heart, and the reason that it is associated with myocarditis and dilated cardiopathy is unknown. Both enteroviruses and Ads can infect cardiac myocytes in culture. It is of interest that coxsackieviruses and Ads both use CAR as the receptor to enter cells. CAR expression in healthy and diseased hearts is reported to be variable, and it has been suggested that this variability plays a role in enterovirus and Ad myocarditis.⁵⁷⁰

A number of reports have diagnosed Ad in myocarditis and dilated myocardiopathy. In a study of 126 conscripts diagnosed on the basis of serial electrocardiograph changes during an acute infection, Ad-specific antibodies increased in 19 patients, although most did not have myocarditis.³⁶² Ad DNA has been detected by PCR in myocardial biopsies primarily from children with acute myocarditis in three studies (see³⁰⁸). Although there was clinical and electrocardiogram evidence of myocarditis, most of the Ad-positive biopsies did not show classic inflammation, in contrast to those that appeared to be associated with enterovirus infection. In children with myocarditis or dilated cardiomyopathy, Ad was detected in anywhere from 8.1% to 36% of patients.⁶⁷,⁶⁸,⁸³,³⁹⁷,⁶²¹,⁶²⁶,⁶⁵⁷ Sequencing of the PCR products from one of these studies established that 80% of the Ad isolates were Ad2; most of the remainder were Ad5 and one was Ad6; nonfatal cases caused by Ad1 have also been described.⁶²¹ In another study of 142 Ad-positive patients with clinical diagnosis of myocarditis, 57 (40%) had histopathology that was consistent with myocarditis; none of the control patients were positive for Ad.⁶⁷ Some patients with detected virus remain positive for a prolonged period of time (mean of 6.8 months); clearance of the virus was associated with improved symptoms.³⁹⁷ Myocardial involvement with Ad may
be patchy, suggesting that biopsies may miss affected regions and thereby underestimate the incidence of Ad-associated disease.²⁶⁸ These studies suggest that Ad may be a cause of or a contributing factor for cardiomyopathy.