A first report of non-invasive adenovirus detection in wild Assamese macaques in Thailand

Several simian adenoviruses (AdVs) have been detected and isolated in various species of non-human primates with the goals of monitoring the health of wildlife and investigating their potential for zoonotic disease transmission. Here, we provide evidence of AdV infection in wild Assamese macaques (Macaca assamensis assamensis) at Phu Khieo Wildlife Sanctuary, Thailand, based on polymerase chain reaction of non-invasively collected fecal samples. Eight out of 110 fecal samples (7.3%), or five out of 87 monkeys (5.7%), showed evidence of AdV infection. All infected individuals were infants or juveniles. Phylogenetic analysis based on the sequence of hexon and polymerase genes revealed two different AdV genotypes. One genotype clustered in the human AdV-G group, while another showed 100% identity with previously reported AdVs of captive Chinese rhesus macaques (Macaca mulatta), which may be tentatively classified as a new species of AdV in non-human primates while awaiting further supporting evidence.     

Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Investigation of adenovirus occurrence in pediatric tumor entities

Adenoviruses (AdVs) contain genes coding for proteins with transforming potential, and certain AdV serotypes have been shown to induce tumors in rodents. However, data on the possible oncogenicity of AdVs in humans are scarce. We have therefore employed a real-time quantitative PCR (RQ-PCR) assay permitting highly sensitive detection of all 51 currently known human AdV serotypes to screen more than 500 tumor specimens derived from 17 different childhood cancer entities including leukemias, lymphomas, and solid tumors. Most tumor entities analyzed showed no evidence for the presence of AdV sequences, but AdV DNA was detected by RQ-PCR in different brain tumors including 25/30 glioblastomas, 22/30 oligodendrogliomas, and 20/30 ependymomas. Nonmalignant counterparts of AdV-positive brain tumors, including specimens of ependymal cells, plexus choroideus, and periventricular white matter, were screened for control purposes and revealed the presence of AdV DNA in most specimens tested. Identification of the AdV types present in positive malignant and nonmalignant brain tissue specimens revealed predominantly representatives of species B and D and, less commonly, C. To exclude contamination as a possible cause of false-positive results, specimens with AdV sequences detectable by PCR were subsequently analyzed by in situ hybridization, which confirmed the PCR findings in all instances. The central nervous system appears to represent a common site of AdV infection with virus persistence, thus providing the first evidence for the possible contribution of AdVs to the multistep process of tumor pathogenesis in brain tissue.     

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.     

Comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications

The adenovirus (AdV) hexon constitutes the major virus capsid protein. The epitopes located on the hexon protein are targets of neutralizing antibodies in vivo, serve in the recognition by cytotoxic T cells, and provide the basis for the classification of adenoviruses into the 51 serotypes known to date. We have sequenced the entire hexon gene from human serotypes with incomplete or no sequence information available (n = 34) and performed a comparative analysis of all sequences. The overall sequence divergence between the 51 human serotypes ranged from 0.7 to 25.4% at the protein level. The sequence information has been exploited to assess the phylogeny of the adenovirus family, and protein distances between the six AdV species (A to F) and among individual serotypes within each species were calculated. The analysis revealed that the differences among serotypes within individual species range from 0.3 to 5.4% in the conserved regions (765 amino acids [aa]) and from 1.5 to 59.6% in the variable regions (154 to 221 aa). Serotypes of different species showed an expectedly greater divergence both in the conserved (5.9 to 12.3%) and variable (49.0 to 74.7%) regions. Construction of a phylogenetic tree revealed three major clades comprising the species B+D+E, A+F, and C, respectively. For serotypes 50 and 51, the original assignment to species B and D, respectively, is not in accordance with the hexon DNA and protein sequence data, which placed serotype 50 within species D and serotype 51 within species B. Moreover, the hexon gene of serotype 16, a member of species B, was identified as the product of recombination between sequences of species B and E. In addition to providing a basis for improved molecular diagnostics and classification, the elucidation of the complete hexon gene sequence in all AdV serotypes yields information on putative epitopes for virus recognition, which may have important implications for future treatment strategies permitting efficient targeting of any AdV serotype.     

Co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in Japan

Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients coinfected with AdV were indistinct from those of patients mono-infected with hMPV. The symptoms of the one patient co-infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa.     

Host Range,Prevalence, and Genetic Diversity of Adenoviruses in Bats

Bats are the second largest group of mammals on earth and act as reservoirs of many emerging viruses. In this study, a novel bat adenovirus (AdV) (BtAdV-TJM) was isolated from bat fecal samples by using a bat primary kidney cell line. Infection studies indicated that most animal and human cell lines are susceptible to BtAdV-TJM, suggesting a possible wide host range. Genome analysis revealed 30 putative genes encoding proteins homologous to their counterparts in most known AdVs. Phylogenetic analysis placed BtAdV-TJM within the genus Mastadenovirus, most closely related to tree shrew and canine AdVs. PCR analysis of 350 bat fecal samples, collected from 19 species in five Chinese provinces during 2007 and 2008, indicated that 28 (or 8%) samples were positive for AdVs. The samples were from five bat species, Hipposideros armiger, Myotis horsfieldii, M. ricketti, Myotis spp., and Scotophilus kuhlii. The prevalence ranged from 6.25% (H. armiger in 2007) to 40% (M. ricketti in 2007). Comparison studies based on available partial sequences of the pol gene demonstrated a great genetic diversity among bat AdVs infecting different bat species as well as those infecting the same bat species. This is the first report of a genetically diverse group of DNA viruses in bats. Our results support the notion, derived from previous studies based on RNA viruses (especially coronaviruses and astroviruses), that bats seem to have the unusual ability to harbor a large number of genetically diverse viruses within a geographic location and/or within a taxonomic group.Members of the family Adenoviridae are nonenveloped, icosahedral viruses approximately 70 to 100 nm in size. The family is divided into four genera: Mastadenovirus, Aviadenovirus, Atadenovirus, and Siadenovirus (3, 6, 7). Adenoviruses (AdVs) contain a linear, nonsegmented, double-stranded DNA (dsDNA) with a genome size ranging from 30 to 36 kb for mastadenoviruses, 31 to 36 kb for atadenoviruses, and 26 to 45 kb for siadenoviruses (3).AdV infection can be identified in mammals, birds, amphibians, reptiles, and fish, and live AdVs have been isolated from at least 40 vertebrate species (3, 6, 21, 25). A total of 52 human AdV (hAdV) serotypes have been identified and classified into seven groups, designated serotypes A through G. AdVs are highly prevalent in the human population and can cause human infections ranging from respiratory disease (mainly by AdV-B and -C) and conjunctivitis (AdV-B and -D) to gastroenteritis (AdV-F serotypes 40 and 41) (11, 24). In animals, canine AdV type 1 (CAV-1) and canine AdV type 2 (CAV-2) cause hepatitis and respiratory and enteric diseases in dogs (20, 30). The egg drop syndrome-1976 virus (EDS-76 virus), belonging to the aviadenoviruses, is the causative agent of an economically important disease characterized by a severe and sudden drop in egg production (17).Bats are reservoirs of numerous new or emerging viruses, including henipavirus, Ebola virus, Marbourg virus, Menangle virus, rabies virus, coronavirus, and astrovirus, and most of the bat viral species reported to date are RNA viruses (4, 5, 14, 23, 28, 31). Although numerous virus species and strains were identified in recent years by PCR and sequencing, the isolation of live bat viruses remains rare and difficult, probably due to the lack of appropriate bat cell lines. Recently, two bat adenoviruses (bat AdV-FBV1 and bat AdV-2 PPV1) were isolated from fruit bat (Pteropus dasymallus yayeyamae) and common pipistrelles (Pteropus pipistrellus), respectively. The agent was identified as novel adenovirus by partial sequencing (16, 22).In this study, we report the isolation of a novel AdV from bat fecal samples using a newly established bat primary kidney cell culture. The isolated AdV, named bat adenovirus strain TJM (BtAdV-TJM), is capable of infecting several vertebrate cell lines and inducing a cytopathic effect (CPE). The near-full-length genome sequence (except the 5′- and/or 3′-terminal ends) of BtAdV-TJM is 31,681 bp and carries 30 putative genes. Our epidemiological investigation demonstrated that among the 19 bat species surveyed in this study, bat AdVs are prevalent mainly in Myotis species and Scotophilus kuhlii. This report represents a first detailed study of a DNA virus group in bats.     

Adenovirus in Rural Côte D`Ivoire: High Diversity and Cross-Species Detection

The Taï region in Western Côte d`Ivoire is characterized by extensive overlap of human and animal habitats. This could influence patterns of adenovirus transmission between humans and domestic animals. Fecal samples from humans and various domestic animals were tested for the presence of adenoviruses by PCR. Phylogenetic and species delineation analyses were performed to further characterize the adenoviruses circulating in the region and to identify potential cross-species transmission events. Among domestic animals, adenovirus shedding was frequent (21.6% of domestic mammals and 41.5% of chickens) and the detected strains were highly diverse, several of them representing novel types. Although no evidence for zoonotic transmission of animal adenovirus was obtained, the present study provides concordant evidence in favor of common cross-species transmission of adenoviruses between different animal species and first indications for adenovirus transmission from humans to animals. These findings underline the thus far underestimated importance of reverse zoonotic transmission of viruses and of the role of domestic animals as pathogen reservoirs, “bridge species,” or intermediate hosts.     

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.     

Retargeting the coxsackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.     

Cryoelectron microscopy map of Atadenovirus reveals cross-genus structural differences from human adenovirus

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.     

TNF-alpha receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-alpha and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. beta-Galactosidase expression in mice receiving intratracheal instillation of Adv/beta-gal (adenovirus construct expressing beta-galactosidase) was transient (less than 14 days), but a significant early increase of beta-galactosidase expression was seen in mice lacking either or both TNF-alpha receptors. Absence of TNF-alpha or the p55 receptor significantly attenuated the Ab response to both adenovirus and beta-galactosidase. Human IL-10 expression in the lung suppressed local TNF-alpha production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with beta-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-alpha signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with beta-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with beta-galactosidase, and is nonimmunogenic in the lung.     

Adenoviruses in medicine: innocuous pathogen,predator, or partner

The consequences of human adenovirus (HAdV) infections are generally mild. However, despite the perception that HAdVs are harmless, infections can cause severe disease in certain individuals, including newborns, the immunocompromised, and those with pre-existing conditions, including respiratory or cardiac disease. In addition, HAdV outbreaks remain relatively common events and the recent emergence of more pathogenic genomic variants of various genotypes has been well documented. Coupled with evidence of zoonotic transmission, interspecies recombination, and the lack of approved AdV antivirals or widely available vaccines, HAdVs remain a threat to public health. At the same time, the detailed understanding of AdV biology garnered over nearly 7 decades of study has made this group of viruses a molecular workhorse for vaccine and gene therapy applications.     

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses’ role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.     

Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.     

Adenoviruses C in non-hospitalized Mexican children older than five years of age with acute respiratory infection

Adenoviruses (AdV) are commonly involved in acute respiratory infections (ARI), which cause high morbidity and mortality in children. AdV are grouped in six species (A-F), which are associated with a wide range of diseases. The aim of this study was to identify the AdV species infecting non-hospitalized Mexican children with ARI symptoms, attending to the same school. For that, a PCR/RFLP assay was designed for a region of the hexon gene, which was chosen, based on the bioinformatical analysis of AdV genomes obtained from GenBank. A total of 100 children's nasopharyngeal samples were collected from January to June, 2005, and used for viral isolation in A549 cells and PCR/RFLP analysis. Only 15 samples produced cytopathic effect, and in all of them AdV C was identified. AdV C was also identified in eight additional nasopharyngeal samples which were negative for viral isolation. In summary, this outpatient population showed a rate of AdV infection of 23%, and only AdV C was detected.     

New adenovirus species found in a patient presenting with gastroenteritis

An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible. Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with a high homology to human adenovirus 41 (HAdV-41) and simian adenovirus 1 (SAdV-1). However, using anti-SAdV-1 sera, it was determined that this virus was serologically different than SAdV-1. Genomic sequencing and phylogenetic analysis confirmed that this new adenovirus was so divergent from the known human adenoviruses that it was not only a new type but also represented a new species (human adenovirus G). In a retrospective clinical study, this new virus was detected by PCR in one additional patient from a separate gastroenteritis outbreak. This study suggests that HAdV-52 may be one of many agents causing gastroenteritis of unknown etiology.