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Adenovirus infection in pediatric liver transplant recipients.

A retrospective review of adenoviral infection in pediatric liver transplant recipients was done at Children's Hospital of Pittsburgh to define its epidemiology and clinical importance. Medical records of patients with adenovirus were reviewed and data collected regarding clinical course, microbiologic studies, biopsy results, immunosuppression, concurrent infections, and outcome. Of 484 liver transplant recipients, 49 had 53 episodes of adenoviral infection. The most common sites of adenoviral infection were the liver, lung, and gastrointestinal tract. Serotypes 1, 2, and 5 were recovered most often; type 5 was commonly associated with hepatitis. Invasive adenoviral infection occurred in 20 children, leading to death in 9. Median time from transplantation until isolation of adenovirus was 25.5 days. This timing suggests either reactivation or donor-associated transmission. Prospective studies using molecular epidemiologic techniques will be helpful in evaluating transmission patterns of adenovirus in this population.

Adenovirus Infections, Human↗

Adenovirus infection inhibits the phosphorylation of major histocompatibility complex class I proteins.

Major histocompatibility complex (MHC) class I molecules act as peptide receptors to direct the recognition of foreign antigens by cytolytic T cells. The cell surface expression and trafficking of these peptide receptors is thought to be controlled by the conformation of the MHC molecule and possibly by the phosphorylation of the cytoplasmic portion of the heavy chain protein. It is of some interest that adenoviruses (Ads) have evolved proteins that interfere with the expression of MHC molecules. One of these proteins, called E3/19k, binds to newly synthesized MHC molecules in the rough endoplasmic reticulum (RER) and inhibits their trafficking to the cell surface. Here we show that during the infection of a human cell line with Ad2, the phosphorylation of the endogenous MHC molecules is inhibited. We also observe that the phosphorylation of the endogenous HLA molecules is grossly impaired in a human cell line transfected with the Ad2 EcoRI D fragment containing the E3/19k gene. We conclude that the E3/19k protein inhibits the phosphorylation of the MHC heavy chains and that this may be one of the important functions of this protein in infected cells. In addition, we show that a mutant of the E3/19k protein, which lacks an RER retention signal but which retains its ability to bind to HLA molecules, does not inhibit the phosphorylation of HLA molecules and that phosphorylated molecules are not Endo H sensitive. This suggests that HLA molecules are phosphorylated after leaving the medial-Golgi compartment, thus providing the most compelling evidence yet that HLA molecules are phosphorylated at or near the cell surface. Finally, to our knowledge, this is the first study under which the phosphorylation of MHC molecules is shown to be altered and may have some relevance for other pathogenic conditions.

Adenoviridae Infections↗

Dependence of adenovirus infectivity on length of the fiber shaft domain.

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/alphav integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/alphav integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and alphav integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.

Adenoviruses, Human↗

In vitro replication of adeno-associated virus DNA: enhancement by extracts from adenovirus-infected HeLa cells.

Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstructural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine replication of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is 50-fold greater in extracts from Ad-infected cells than in extracts from uninfected cells. In addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated strands, whereas the majority of labeled full-length AAV DNA molecules synthesized in the uninfected extract have only one newly replicated strand. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the case of the Ad-infected cell extract is much more likely to result in the synthesis of a full-length AAV DNA molecule. Most of the newly replicated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provides a helper function related to elongation during replication by a single-strand displacement mechanism. In the assay using the uninfected HeLa cell extract, replication frequently stalls before reaching the end of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template. In support of this conjecture, replication in the uninfected cell extract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA. In addition, when shorter substrate molecules are used as the template in the uninfected HeLa cell assay, a greater proportion of the labeled full-length substrate molecules contain two newly replicated strands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad-infected cells.

Cell Extracts↗

Adenovirus infection of a renal allograft.

One month after renal transplantation, a 60-year-old man developed acute allograft dysfunction associated with gross hematuria and dysuria. Urinary cytological examination showed viral inclusion-bearing epithelial cells. A renal transplant biopsy specimen showed granulomatous interstitial nephritis, tubular necrosis, and ground glass-like intranuclear viral inclusion bodies in tubular cells caused by an adenovirus (ADV) infection. A reduction in baseline immunosuppressive therapy resulted in rapid normalization of allograft function and ultimately viral clearance. We report this case not only to illustrate an exceptional manifestation of an ADV infection in a renal allograft, but also to highlight the beneficial effect of reduction in immunosuppressive therapy on viral replication and clinical outcome.

Acute Kidney Injury↗

The E4-6/7 protein functionally compensates for the loss of E1A expression in adenovirus infection.

The E1A gene products are required and sufficient for activation of adenovirus gene expression in cultured cells. The E4-6/7 gene product induces the binding of the cellular transcription factor E2F to the viral E2a promoter region. The induction of E2F binding to the E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter in vivo. The E2 region encodes the viral replication proteins, yet adenoviruses lacking E4-6/7 function demonstrate no defective phenotype in infected cells. Here we show that the E4-6/7 protein can functionally compensate for E1A expression in virus infection. In the absence of the E1A gene products, expression of the E4-6/7 protein is sufficient to displace retinoblastoma protein family members from E2Fs, activate expression of early region 2 via induction of E2F DNA binding to the E2a promoter region, and significantly enhance replication of an E1A-defective adenovirus. These results have implications in the regulation of viral gene expression and for the development of recombinant adenovirus vectors.

Adenovirus E1A Proteins↗

Alveolar macrophages and T cells from sarcoid, but not normal lung, are permissive to adenovirus infection and allow analysis of NF-kappa b-dependent signaling pathways.

Adenovirus (Adv)-mediated gene transfer requires efficient infection of target cells. The objective of this study was to establish whether alveolar macrophages (AM) and T cells (AT) from sarcoid patients were permissive to infection with Adv vectors and if this property could be used to investigate cytokine gene regulation. Sarcoid and normal bronchoalveolar lavage (BAL) specimens infected with Adv vectors expressing either beta-galactosidase or a green fluorescent protein were analyzed for transgene expression by fluorescence-activated cell sorter (FACS) and direct immunofluorescence, respectively. Expression of surface antigens previously associated with Adv infection, the coxsackie/adenovirus receptor (CAR), alpha v beta 3, and alpha v beta 5 integrins, was also assessed using FACS analysis. Sarcoid AM and AT were found to efficiently express Adv transgenes, unlike AM from normal volunteers, peripheral blood monocytes, and peripheral blood T cells. Cells permissive to Adv infection expressed the CAR and alpha v beta 5 integrin (also alpha v beta 3 integrin for AM). The data indicate that the upregulation of Adv receptors and the ability to infect sarcoid AM and AT are related to the inflammatory environment within the lung. Having demonstrated efficient Adv-mediated transgene delivery to sarcoid AM and AT, a construct encoding porcine I kappa B alpha was then used to investigate the requirement for nuclear factor (NF)-kappa B in the regulation of cytokine gene expression in pulmonary sarcoidosis. Overexpression of I kappa B alpha in sarcoid BAL specimens indicated that tumor necrosis factor-alpha and interleukin (IL)-6 production by AM and interferon (IFN)-gamma production by AT is NF-kappa B dependent, whereas IL-4 production by AT is NF-kappa B independent. This is the first occasion that the requirement for NF-kappa B in IFN-gamma gene expression within primary human T cells has been demonstrated. The results of this study have implications for the future investigation of molecular pathways in inflammatory lung disease.

Adenoviridae↗

Neonatal pulmonary insufficiency caused by adenovirus infection successfully treated with extracorporeal membrane oxygenation.

Two infants with fulminant early-onset sepsis syndrome and respiratory failure are described. Adenovirus was isolated from cultures from both patients. Complications during pregnancy and respiratory failure that required tracheal intubation at birth suggested congenital infection. Both infants were successfully treated with extracorporeal membrane oxygenation.

Adenovirus Infections, Human↗

Neutralization of adenovirus infectivity and cytotoxin in various cell cultures.

The neutralization of human adenovirus 5 and 11 by homologous and heterologous rabbit antisera was determined by CPE inhibition in various cell cultures (HeLa, HEL, Vero, secondary kidney cells from cercopithecus, rabbit, mouse), or in HeLa cells made impermissive by IUdR inhibition. The results concerning sensitivity and specificity were similar in all cases. Crude and purified virus showed similar neutralization. Immunofluorescence neutralization in HeLa cell cultures gave similar results; this method is suitable for demonstrating subtle immunological relations between adenovirus types. The neutralization of the early cytopathic factor ('cytotoxin') showed a pattern of cross-reactivity different from the virion; the cytotoxin was found to be active in part of the cell cultures only. It is concluded from the results that the virus function(s) blocked by antibody appear to be identical for the replicative cycle in infection and for the initiation of the abortive infection in non-permissive cells. Hence, either kind of cells may be used for neutralization tests.

Adenoviruses, Human↗

Enteric adenovirus infection and childhood diarrhea: an epidemiologic study in three clinical settings.

During a 2-year prospective study of gastroenteritis in children less than 2 years of age, the role of enteric adenovirus as a cause of infantile diarrhea was examined in three clinical settings in a case-control fashion. Using a monoclonal antibody-based enzyme-linked immunosorbent assay with specificity for adenovirus serotypes 40 and 41, enteric adenovirus was identified in 10 of 246 episodes of diarrhea in outpatients (4.1%), 13 of 211 children admitted to the hospital with diarrhea (6.2%), and 5 of 81 children in whom nosocomial diarrhea developed (6.2%), making this agent the third most commonly identified etiologic agent of diarrheal disease. Asymptomatic infections were uncommon (5 of 372 control subjects, or 1.3%) and were seen most frequently in the nosocomial setting. Cases occurred in every calendar month except March and April of each year. A syndrome of watery diarrhea of longer duration compared with other patients with diarrhea (mean 5.4 vs 3.8 days, P = .01), associated with vomiting and dehydration, was present in most cases. Compared with patients with rotavirus, patients were as likely to experience fever and dehydration and more likely to vomit. Household contact with gastroenteritis, often with a child 2 to 5 years of age, was a predisposing factor. It was concluded that enteric adenovirus is an important cause of infantile diarrhea in Baltimore children. Although far less common than rotavirus, this agent was associated with diarrheal illnesses that were at least as severe as those seen with rotavirus.

Adenoviridae Infections↗

Cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation.

Adenovirus is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation and there is no established therapy. Cidofovir has in vitro efficacy against adenovirus. We performed a retrospective analysis of 45 patients treated with cidofovir for adenovirus from 10 centers. In total, 16 patients had definite adenovirus disease, 13 probable disease, and 16 asymptomatic infections. A total of 31 (69%) patients were successfully treated with cidofovir, 10 failed, and four were not evaluable owing to early death from other causes. Cidofovir therapy was successful in 10 patients with adenovirus disease, 10 patients with probable disease, and in 10 patients with asymptomatic infections. The overall survival at 28 days and 6 months after initiation of cidofovir therapy was 76 and 46%, respectively. Of the patients, 18 developed toxicity associated with cidofovir: 14 developed renal toxicity and four other types of toxicities. We conclude that cidofovir may be useful against adenovirus after allogeneic hematopoietic stem cell transplantation but additional studies are needed.

Adenovirus Infections, Human↗