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Purpose Outbreaks of adenovirus in neonatal intensive care units (NICUs) can lead to widespread transmission and serious adverse outcomes. We describe the investigation, response, and successful containment of an adenovirus outbreak in a NICU associated with contaminated handheld ophthalmologic equipment used during retinopathy of prematurity (ROP) screening. Design Epidemiologic outbreak investigation. Participants A total of 23 hospitalized neonates, as well as NICU staff and parents of affected infants. Main Outcome Measures Routine surveillance identified an adenovirus outbreak in a level IV NICU in August 2016. Epidemiologic investigation followed, including chart review, staff interviews, and observations. Cases were defined as hospital-acquired adenovirus identified from any clinical specimen (NICU patient or employee) or compatible illness in a family member. Real-time polymerase chain reaction (PCR) and partial- and whole-genome sequencing assays were used for testing of clinical and environmental specimens. Results We identified 23 primary neonatal cases and 9 secondary cases (6 employees and 3 parents). All neonatal case-patients had respiratory symptoms. Of these, 5 developed pneumonia and 12 required increased respiratory support. Less than half (48%) had ocular symptoms. All neonatal case-patients (100%) had undergone a recent ophthalmologic examination, and 54% of neonates undergoing examinations developed adenovirus infection. All affected employees and parents had direct contact with infected neonates. Observations revealed inconsistent disinfection of bedside ophthalmologic equipment and limited glove use. Sampling of 2 handheld lenses and 2 indirect ophthalmoscopes revealed adenovirus serotype 3 DNA on each device. Sequence analysis of 16 neonatal cases, 2 employees, and 2 lenses showed that cases and equipment shared 100% identity across the entire adenovirus genome. Infection control interventions included strict hand hygiene, including glove use; isolation precautions; enhanced cleaning of lenses and ophthalmoscopes between all examinations; and staff furlough. We identified no cases of secondary transmission among neonates. Conclusions Adenovirus outbreaks can result from use of contaminated ophthalmologic equipment. Even equipment that does not directly contact patients can facilitate indirect transmission. Patient-to-patient transmission can be prevented with strict infection control measures and equipment cleaning. Ophthalmologists performing inpatient examinations should take measures to avoid adenoviral spread from contaminated handheld equipment.

Nuclear import of viral genomes is an important step during the life cycle of adenoviruses (AdV), requiring soluble cellular factors as well as proteins of the nuclear pore complex (NPC). We addressed the role of the cytoplasmic nucleoporin Nup358 during adenoviral genome delivery by performing depletion/reconstitution experiments and time-resolved quantification of adenoviral genome import. Nup358-depleted cells displayed reduced efficiencies of nuclear import of adenoviral genomes, and the nuclear import receptor transportin 1 became rate limiting under these conditions. Furthermore, we identified a minimal N-terminal region of Nup358 that was sufficient to compensate for the import defect. Our data support a model where Nup358 functions as an assembly platform that promotes the formation of transport complexes, allowing AdV to exploit a physiological protein import pathway for accelerated transport of its DNA. IMPORTANCE Nuclear import of viral genomes is an essential step to initiate productive infection for several nuclear replicating DNA viruses. On the other hand, DNA is not a physiological nuclear import substrate; consequently, viruses have to exploit existing physiological transport routes. Here, we show that adenoviruses use the nucleoporin Nup358 to increase the efficiency of adenoviral genome import. In its absence, genome import efficiency is reduced and the transport receptor transportin 1 becomes rate limiting. We show that the N-terminal half of Nup358 is sufficient to drive genome import and identify a transportin 1 binding region. In our model, adenovirus genome import exploits an existing protein import pathway and Nup358 serves as an assembly platform for transport complexes.

Adenovirus is a common human pathogen that relies on host cell processes for production and processing of viral RNA. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. The canonical viral early and late RNA cassettes were confirmed, but analysis of splice junctions within long RNA reads revealed an additional 20 novel viral transcripts. These RNAs include seven new splice junctions which lead to expression of canonical open reading frames (ORF), as well as 13 transcripts encoding for messages that potentially alter protein functions through truncations or the fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

BACKGROUND Achalasia, as an incurable disease is defined by the lack of normal esophageal peristalsis and loss of lower esophageal sphincter relaxation due to impaired myenteric neural plexus. The exact cause of myenteric neural cells degeneration in achalasia is still unknown. One hypothesis is that certain neurotropic viruses and autoimmune factors cause the inflammatory response in myenteric network, which consequently destroy neural cells. This study was designed to find the evidence of viral causes of achalasia. METHODS In this case-control study, 52 patients with achalasia and 50 controls referred to Shariati Hospital, were evaluated for the genome of neurotropic viruses, HPV, and adenovirus by polymerase chain reaction (PCR) and reverse transcription (RT) PCR techniques. RESULTS Genome assessment of neurotropic DNA viruses turned out negative in the patients, however, the genome of HSV-1 (Herpes simplex virus) was found in tissues of six controls. No neurotropic RNA viruses were observed in the tissue samples and whole blood of both the patients and controls. Among non-neurotropic viruses, adenovirus genome was positive in tissues of two out of 52 patients and three out of 50 controls. In addition, one out of 52 patients and two out of 50 controls were positive for HPV infection in tissues. CONCLUSION We could not detect any significant relationship between achalasia and HPV, adenovirus, and neurotropic viruses in the cases. Nevertheless, it does not exclude the hypothesis of either an alternate viral species or resolved viral infection as the etiology of achalasia.

Recently, human adenovirus type 3 (HAdV-3) has become the most isolated HAdV worldwide. Restriction endonuclease analysis of globally isolated strains of HAdV-3 has uncovered 51 genome types to date. Information on the genome type is important to the epidemiological study of HAdV-3. In this study, analysis of 75 isolates of HAdV- 3 collected over a 24-year period in Fukui revealed: (1) the emergence of three novel genome types (HAdV-3a52, HAdV-3a53 and HAdV-3a54) and two known genome types (HAdV-3a and HAdV-3a54); (2) the spectrum of diseases caused by individual genome types and their major involvement in the paediatric age population; and (3) the co-circulation and replacement of genome types as a usual phenomenon. The rising number of HAdV-3 genome types indicates that the genetic variation of HAdV-3 is more than other HAdVs. Considering the clinical importance of HAdV-3 infection, its genetic diversity underscores the need for its continuous surveillance and genetic characterization.

Adenoviruses are common pathogens in vertebrates, infecting a wide range of hosts, but only having rarely been detected and correlated with disease in cetaceans. This article describes the first complete genomic sequence of a cetacean adenovirus, bottlenose dolphin adenovirus 1 (BdAdV-1), detected in captive bottlenose dolphin population (Tursiops truncatus) suffering from self-limiting gastroenteritis. The complete genome sequence of BdAdV-1 was recovered from data generated by high-throughput sequencing and validated by Sanger sequencing. The genome is 34,080bp long and has 220 nucleotides long inverted terminal repeats. A total of 29 coding sequences were identified, 26 of which were functionally annotated. Among the unusual features of this genome is a remarkably long 4380bp E3 ORF1, that displays no sequence homology with the corresponding E3 regions of other adenoviruses. In addition, the fiber protein only has 26% identity with fiber proteins described in other adenoviruses. Three hypothetical proteins were predicted. The phylogenetic analysis indicates that the closest known relative to BdAdV-1 is an adenovirus detected in bottlenose dolphin (KR024710), with an amino acid sequence identity between 36 and 79% depending on the protein. Based on the phylogenic analysis, the BdAdV-1 appears to have co-evolved with its host. The results indicate that BdAdV-1 belongs to the Mastadenovirus genus of the Adenoviridae family, however, it is clearly different from other adenoviruses, especially in the 3'-end of the viral genome. The high degree of sequence divergence suggests that BdAdV-1 should be considered as a novel species in the Mastadenovirus genus. The study also demonstrates the usefulness of high-throughput sequencing to obtain full-length genomes of genetically divergent viruses.

Adenoviruses are widely used to deliver genes to a variety of cell types and have been used in a number of clinical trials for gene therapy and oncolytic virotherapy. However, several concerns must be addressed for the clinical use of adenovirus vectors. Selective delivery of a therapeutic gene by adenovirus vectors to target cancer is precluded by the widespread distribution of the primary cellular receptors. The systemic administration of adenoviruses results in hepatic tropism independent of the primary receptors. Adenoviruses induce strong innate and acquired immunity in vivo. Furthermore, several modifications to these vectors are necessary to enhance their oncolytic activity and ensure patient safety. As such, the adenovirus genome has been engineered to overcome these problems. The first part of the present review outlines recent progress in the genetic modification of adenovirus vectors for cancer treatment. In addition, several groups have recently developed cancer‐targeting adenovirus vectors by using libraries that display random peptides on a fiber knob. Pancreatic cancer‐targeting sequences have been isolated, and these oncolytic vectors have been shown by our group to be associated with a higher gene transduction efficiency and more potent oncolytic activity in cell lines, murine models, and surgical specimens of pancreatic cancer. In the second part of this review, we explain that combining cancer‐targeting strategies can be a promising approach to increase the clinical usefulness of oncolytic adenovirus vectors.

Competing roles of coevolution, selective pressure and recombination are an emerging interest in virus evolution. We report a novel aviadenovirus from captive red-bellied parrots (Poicephalus rufiventris) that uncovers evidence of deep recombination among aviadenoviruses. The sequence identity of the virus was most closely related to Turkey adenovirus D (42% similarity) and other adenoviruses in chickens, turkeys and pigeons. Sequencing and comparative analysis showed that the genome comprised 40,930 nucleotides containing 42 predicted open reading frames (ORFs) 19 of which had strong similarity with genes from other adenovirus species. The new genome unveiled a lineage that likely participated in deep recombination events across the genus Aviadenovirus accounting for an ancient evolutionary relationship. We hypothesize frequent host switch events and recombination among adenovirus progenitors in Galloanserae hosts caused the radiation of extant aviadenoviruses and the newly assembled Poicephalus adenovirus genome points to a potentially broader host range of these viruses among birds.

Background Human adenoviruses (HAdVs) are known causes of respiratory illness outbreaks in congregate settings, but cases and clusters are less well described from community settings in the United States. During December 2016–February 2017, the New Jersey Department of Health received reports of HAdV infections from 3 sources in 3 adjacent counties. We investigated to characterize the epidemiologic, laboratory, and clinical features of this HAdV outbreak. Methods A case was defined as a New Jersey resident with acute respiratory illness during December 1, 2016–March 31, 2017 with laboratory identification of HAdV genome type 7d (HAdV-7d). Human adenovirus was detected by real-time and conventional polymerase chain reaction and molecular typed by partial hexon capsid protein gene sequencing. The HAdV genome type was identified by whole genome sequencing analysis. Available medical, public health, and surveillance records were reviewed. Results We identified 12 cases, including 3 treatment facility patients, 7 college students, and 2 cases at a tertiary-care hospital. Four cases died; all had underlying comorbidities. Nine HAdV-7d whole genome sequences obtained from all 3 sites were nearly identical. Conclusions Transmission of HAdV-7d occurred in community and congregate settings across 3 counties and resulted in severe morbidity and mortality in some cases with underlying comorbidities. Clinicians and local and state health departments should consider HAdV in patients with severe respiratory infection.

Adenoviruses cause variety of infections in mammals and birds leading to high morbidity and mortality in immunocompromised hosts. Adenoviruses replicate in hosts cell nucleus and code their own DNA polymerase. The first eukaryotic DNA replication system for which both initiation and elongation could be reconstituted in vitro was that of the adenovirus. Three viral proteins are required for adenovirus genome replication: (1) adenovirus DNA polymerase (Ad pol), (2) the precursor terminal protein (pTP), and (3) the DNA-binding protein (DBP). The chapter presents a detailed account of Ad pol, which is a 140 kDa protein that belongs to protein-primed polymerases of family B (Alpha like) of DNA-dependent DNA polymerases. It is functionally conserved with the polymerase activity located at C terminals and has molecular architecture similar to DNA polymerase of bacteriophage RB69. Ad pol is a processive enzyme and in addition to a polymerase activity also possesses 3ʹ–5ʹ exonuclease activity. In recent years, it has emerged as a promising target for development of diagnostic and therapeutic agents against adenoviral diseases.

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore., Spanish Ministry of Economy and Competitivity [FIS2017-89549-R; “María de Maeztu” Program for Units of Ex-cellence in R&D MDM-2014-0377; and FIS2017-90701-REDT] and Human Frontiers Science Program [HFSPORGP0012/2018] to P.J.P. Spanish State Research Agency and European Regional Development Fund [BFU2016-74868-P] and Spanish Ministry of Economy, Industry and Competitivity [BIO2015-68990-REDT, the Spanish Aden-ovirus Network, AdenoNet] to C.S.M. MINECO/FEDER,UE [FIS2015-67837-P] to D.R. National Institutes ofHealth [CA122677 and AI102577]

The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs.

Bats carry diverse RNA viruses, some of which are responsible for human diseases. Compared to bat-borne RNA viruses, relatively little information is known regarding bat-borne DNA viruses. In this study, we isolated and characterized three novel bat adenoviruses (BtAdV WIV9-11) from Rhinolophus sinicus. Their genomes, which are highly similar to each other but distinct from those of previously sequenced adenoviruses (AdVs), are 37 545, 37 566 and 38 073 bp in size, respectively. An unusually large E3 gene was identified in their genomes. Phylogenetic and taxonomic analyses suggested that these isolates represent a distinct species of the genus Mastadenovirus. Cell susceptibility assays revealed a broad cell tropism for these isolates, indicating that they have a potentially wide host range. Our results expand the understanding of genetic diversity of bat AdVs.

The pathogenesis of intussusception without obvious anatomical leading points remains unclear. The objective of this study was to determine a feasibility of association between certain gastroenteritis viruses and intussusception. This was a prospective cohort study. Forty intussusception cases and 136 acute gastroenteritis controls with comparable age and gender were separately consecutively enrolled and relevant clinical data of both groups were recorded. The clinical specimens collected from all patients were screened for adenovirus, rotavirus, norovirus, and astrovirus by PCR and RT-PCR using specific primers. The genomes of detected viruses were characterized further to identify their genotypes by nucleotide sequencing. In 40 intussusception cases, adenovirus, rotavirus, and norovirus were detected in 12 (30.0%), 2 (5.0%), and 2 (5.0%), respectively while astrovirus was undetectable. In contrast, 136 acute gastroenteritis patients, adenovirus, rotavirus, and norovirus were detected in 11 (8.1%), 24 (17.7%), and 24 (17.7%) patients, respectively and again astrovirus was undetectable. The detection of adenovirus in intussusception patients was significantly higher than those in the control group (P

Recent studies involving several viral systems have highlighted the importance of cellular intrinsic defense mechanisms through nuclear antiviral proteins that restrict viral propagation. These factors include among others components of PML nuclear bodies, the nuclear DNA sensor IFI16, and a potential restriction factor PHF13/SPOC1. For several nuclear replicating DNA viruses, it was shown that these factors sense and target viral genomes immediately upon nuclear import. In contrast to the anticipated view, we recently found that incoming adenoviral genomes are not targeted by PML nuclear bodies. Here we further explored cellular responses against adenoviral infection by focusing on specific conditions as well as additional nuclear antiviral factors. In line with our previous findings, we show that neither interferon treatment nor the use of specific isoforms of PML nuclear body components results in co-localization between incoming adenoviral genomes and the subnuclear domains. Furthermore, our imaging analyses indicated that neither IFI16 nor PHF13/SPOC1 are likely to target incoming adenoviral genomes. Thus our findings suggest that incoming adenoviral genomes may be able to escape from a large repertoire of nuclear antiviral mechanisms, providing a rationale for the efficient initiation of lytic replication cycle.

Cancer therapy using oncolytic viruses is an emerging area, in which viruses are engineered to selectively propagate in tumor tissues without affecting healthy cells. Because of the advantages that adenoviruses (Ads) have over other viruses, they are more considered. To achieve tumor selectivity, two main modifications on Ads genome have been applied: small deletions and insertion of tissue- or tumor-specific promoters. Despite oncolytic adenoviruses ability in tumor cell lysis and immune responses stimulation, to further increase their antitumor effects, genomic modifications have been carried out including insertion of checkpoint inhibitors and antigenic or immunostimulatory molecules into the adenovirus genome and combination with dendritic cells and chemotherapeutic agents. This study reviews oncolytic adenoviruses structures, their antitumor efficacy in combination with other therapeutic strategies, and finally challenges around this treatment approach.

Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota., Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27kb. A 907bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

SummaryIn response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here, we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN-independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate “self” and “non-self” genomes and determine whether a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability.

There is little information about the presence of human adenovirus (HAdV) in drinking water in Neotropical regions. Thus, the present study sought to conduct quantification and molecular characterization of HAdVs detected in treated water samples from an area of the Cerrado ecoregion of Brazil. Between August and November 2012, samples were collected from four treated water reservoirs and their respective sites along the water distribution network of the city of Goiânia, for a total of 80 samples. All samples were concentrated and analyzed by qPCR, and selected samples were sequenced. Overall, 76.6 (10(0)-10(9) GC mL(-1)) and 37.5% (10(1)-10(8) GC mL(-1)) of samples drawn from reservoirs and their distribution sites, respectively, were positive for virus by qPCR. All samples selected for sequencing were characterized as species C human adenovirus. Such high HAdV counts have in treated water samples. This finding merits special attention, particularly from the sanitation authorities, because the high number of GC mL(-1) may be an indicative of risk to human health.

Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector.

Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin. IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases, such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally downregulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome.

A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution.

Bats have been reported to carry diverse adenoviruses. However, most bat adenoviruses have been identified on the basis of partial genome sequences, and knowledge on the evolution of bat adenoviruses remains limited. In this study, we isolated and characterized four novel adenoviruses from two distinct bat species, and their full-length genomes were sequenced. Sequence analysis revealed that these isolates represented three distinct species of the genus Mastadenovirus. However, all isolates had an exceptionally low G+C content and relatively short genomes compared with other known mastadenoviruses. We further analysed the relationships among the G+C content, 5′-C-phosphate-G-3′ (CpG) representation and genome size in the family Adenoviridae. Our results revealed that the CpG representation in adenoviral genomes depends primarily on the level of methylation, and the genome size displayed significant positive correlations with both G+C content and CpG representation. Since ancestral adenoviruses are believed to have contained short genomes, those probably had a low G+C content, similar to the genomes of these bat strains. Our results suggest that bats are important natural reservoirs for adenoviruses and play important roles in the evolution of adenoviruses.

Multiple novel simian adenoviruses have been isolated over the past years and their potential to cross the species barrier and infect the human population is an ever present threat. Here we describe the isolation and full genome sequencing of a novel simian adenovirus (SAdV) isolated from the urine of two independent, never co-housed, late stage simian immunodeficiency virus (SIV)-infected rhesus macaques. The viral genome sequences revealed a novel type with a unique genome length, GC content, E3 region and DNA polymerase amino acid sequence that is sufficiently distinct from all currently known human- or simian adenovirus species to warrant classifying these isolates as a novel species of simian adenovirus. This new species, termed Simian mastadenovirus D (SAdV-D), displays the standard genome organization for the genus Mastadenovirus containing only one copy of the fiber gene which sets it apart from the old world monkey adenovirus species HAdV-G, SAdV-B and SAdV-C.

Adenovirus (Ad) vectors are currently the most commonly used platform for therapeutic gene delivery in human gene therapy clinical trials. Although these vectors are effective, many researchers seek to further improve the safety and efficacy of Ad-based vectors through detailed characterization of basic Ad biology relevant to its function as a vector system. Most Ad vectors are deleted of key, or all, viral protein coding sequences, which functions to not only prevent virus replication but also increase the cloning capacity of the vector for foreign DNA. However, radical modifications to the genome size significantly decreases virion stability, suggesting that the virus genome plays a role in maintaining the physical stability of the Ad virion. Indeed, a similar relationship between genome size and virion stability has been noted for many viruses. This review discusses the impact of the genome size on Ad virion stability and emphasizes the need to consider this aspect of virus biology in Ad-based vector design.

Replication competent adenovirus (RC-Ad) vectors mediate robust transgene expression by virtue of amplifying transgenes by replication but also put patients at a risk of frank adenovirus infection. In contrast, E1-deleted replication defective Ad (RD-Ad) vectors are safer but produce substantially less transgene product. To generate a robust, but safer adenoviral vector, we created a “single cycle” adenovirus (SC-Ad) vector that replicates its genome and transgene, but that does not cause adenovirus infections by deleting the capsid cement protein IIIa in low seroprevalence adenovirus serotype 6. Ad6-ΔIIIa can be produced in IIIa-expressing cell lines. In normal cells, Ad6-ΔIIIa replicates its genome and transgene but fails to package its DNA or form mature virus. SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad in human and mouse cells in vitro and in vivo in mice. These data suggest that SC-Ads may be safer amplifying vectors for vaccine and therapeutic applications.

A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five genome regions. Of these, 62 isolates were HAdV-B3 and HAdV-B7 collected during a simultaneous 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 and other species B adenovirus types collected in different years following the outbreak. The phylogenetic analysis results of all the isolates in the structural regions hexon L2, penton, and fiber knob were found to be consistent and no mismatches were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable even when different types are circulating in a limited geographical area simultaneously. J. Med. Virol. 87: 117–124, 2015. © 2014 Wiley Periodicals, Inc.

After generating the recombinant adenovirus plasmid by direct cloning, the viral genome must be released from the plasmid for virus rescue and expansion in corresponding packaging cell lines. This protocol describes how to process and rescue a recombinant adenovirus and grow the rescued virus to large scale.

Objective To construct a recombinant adenovirus vector carrying hSulf-1 gene and to investigate its effect on the proliferation and migration of human umbilical vein endothelial cells.Methods The hSulf-1 protein was inserted into adenovirus genome to generate recombinant adenovirus Ad5-hSulf1,and then the virus was used to infect ECV-304 cell line.The expression of hSulf-1 protein was detected by Western blotting analysis,the cell viability was examined by MTT assay,and the cell migration ability was evaluated by wound-healing assay in ECV-304 cells.Results The recombinant adenovirus Ad5-hSulf1 was successfully constructed.Western blotting analysis showed that over-expression of hSulf1 down-regulated the phosphorylation levels of Akt and ERK in ECV-304 cells.MTT assay showed that over-expression of hSulf1 inhibited the proliferation of ECV-304 cells,with the cell survival rate decreased to(68.49±0.05)% at MOI of 50 pfu/ml and(67.78±0.06)% at MOI of 100 pfu/ml(P0.05).Wound-healing assay showed that over-expression of hSulf1 significantly inhibited the migration of ECV-304 cells compared with the control group(P0.01).Conclusion The recombinant adenovirus Ad5-hSulf1-mediated hSulf-1 over-expression can markedly inhibit the proliferation and migration of ECV-304 cells,laying a foundation for hSulf-1 related gene therapy of tumor and angiogenesis-associated diseases.

Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber.

The study describes successful isolation of 96 fowl adenovirus (FAdV) strains from 789 chickens from 95 flocks. PCR specific for hexon gene encoding L1 loop was conducted. Amplicons were subjected to sequence analysis. The sequences were analysed by the software: BLAST, Geneious 6.0, and MEGA 5, then aligned with different adenovirus strain reference sequences accessible in GenBank database. The examined strains belong to the particular groups and serotypes. The sequences of all adenoviruses were classified into five species (FAdV A–E) and eight serotypes (FAdV-1, FAdV-2, FAdV-4, FAdV-5, FAdV-7, FAdV-8a, FAdV- 8b, and FAdV-11). Key words: chickens, aviadenoviruses, PCR, phylogenetic analysis, Poland. Introduction Adenoviral infections are widely prevalent among birds including poultry and wild species in almost every country in the world (7). Each year in Poland, the number of cases of adenovirus infections in birds is increasing. The pathogenic role of the aviadenoviruses is not clear enough. They can be isolated from both sick birds, and birds without any signs of illness (12, 16). The role of adenoviruses as the opportunistic factors is indicated (7). They can cause asymptomatic infections, but also they are an aetiological agent of diseases such as inclusion body hepatitis – IBH (6), hydropericardium hepatitis syndrome – HHS (7), gizzard erosion and ulceration (GEU) (20), and pancreatitis (24). Adenoviruses can be one of the reasons of immunodeficiency. Aviadenoviruses are icosahedral in shape and non-enveloped dsDNA viruses with capsid size about 70-100 nm in diameter, which consists of 252 capsomers surrounding the core (13, 16). Hexon is the main protein of the adenovirus capsid, encoded by 2800-2900 bp long fragment, with molecular weight of 106,7 kDa, (1, 4). The protein consists of conservative components P1 and P2, which build the basis of the particle and allow to form trimmers. The variable fragment of the hexon protein forms the loops L1, L2, and, L4 (21). The L1 loop (Hyper Variable Region, HVR) is 130 aa long and flanked by the P1 region (8). There are four HVRs located in loop L1, two HVRs in loop L2, and one in loop L4. The type-specific antigenic determinant „e” is encoded by HVR region of L1 and L2 loops (21), while the sequence of L1 loop of adenovirus genome is specific for 12 serotypes of adenovirus (21). This protein is responsible for the antigenic activity of the virus. Adenoviruses existing in poultry were divided into five species (FAdV A–E) and 12 serotypes (FAdV-1–8a, 8b–11) (5, 16, 23). The aim of this study was the molecular analysis of fowl adenovirus strains on the basis of L1 DNA sequences of hexon gene.

Computational analysis of human adenovirus type 4 (HAdV-E4), a pathogen that is the only HAdV member of species E, provides insights into its zoonotic origin and molecular adaptation. Its genome encodes a domain of the major capsid protein, hexon, from HAdV-B16 recombined into the genome chassis of a simian adenovirus. Genomes of two recent field strains provide a clue to its adaptation to the new host: recombination of a NF-I binding site motif, which is required for efficient viral replication, from another HAdV genome. This motif is absent in the chimpanzee adenoviruses and the HAdV-E4 prototype, but is conserved amongst other HAdVs. This is the first report of an interspecies recombination event for HAdVs, and the first documentation of a lateral partial gene transfer from a chimpanzee AdV. The potential for such recombination events are important when considering chimpanzee adenoviruses as candidate gene delivery vectors for human patients.

Conditionally replicative adenoviruses represent an innovative group of anticancer agents designed to destroy these cells by replication and lysis. A major problem associated with of the use of adenoviral vectors in gene therapy is its high liver uptake and lack of tumor selectivity upon systemic administration. To improve the efficacy of CRAds as anticancer agents, their infection efficiency on CAR-deficient tumor cells could be enhanced their by redirecting viral entry via a CAR-independent pathway. To redirect the entry pathway of adenoviruses and enhance their infectivity and specificity, two general strategies are being used. In the first strategy, the adenovirus genome is changed to alter the binding specificity of the viral capsid. In the second strategy, a two-component targeted adenovirus is created by binding of proteins with specific affinity for cancer cells onto the viral capsid. Despite effective targeting and tumor eradication in vitro and in mouse models, the results from systemic administration of targeted CrAds is limited. In addition, clinical effects of CrAds are disappointing up till now. Therefore, combination therapies in which targeted CrAds are combined with other types of therapy are being investigated.

Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging.

The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system.

Oncolytic viruses are emerging as a promising alternative for cancer therapy. Among these biologic agents, adenoviruses have been extensively tested as vectors to transfer exogenous genes to cancer cells. Sophisticated modifications of the adenovirus genome to improve infectivity and selectivity have made possible the production of replicating adenoviruses intended to directly destroy tumors by cell lysis. In this chapter, we discuss the origins of virotherapy and early trials with wild-type adenoviruses and other viruses, and the concept underlying the development of replication-deficient adenoviral vectors, with special emphasis on Ad-p53. We then review clinical findings for dl1520 and H101 oncolytic adenoviruses and, finally, the concepts of Delta-24, Delta-24-RGD, and ICOVIR, emphasizing clinical applications. A dramatic increment in clinical trials over the next 5 years will lead to the approval of oncolytic viruses for conventional cancer treatment within the next decade.

Adenoviral vectors have yielded promising results as carriers for gene transfer and vaccines in basic research and clinical applications. However, most common procedures to construct adenoviral vectors and manipulate adenovirus (Ad) genomes are complex and labor-intensive. An easy and detailed protocol for the rapid, efficient, and modular generation of chimpanzee Ad serotype 68 (AdC68) as a molecular clone via isothermal assembly, which directionally assembles multiple DNA fragments in a single isothermal reaction without restriction enzymes or ligases, is presented. Any serotype of adenovirus with the sequence of genome known can be constructed as a molecular clone by this method. Recombinant adenoviral vectors can be created via one-step isothermal assembly in