Genome analysis of a novel avian atadenovirus reveals a possible horizontal gene transfer.

We report the discovery and characterization of a novel adenovirus, Zoothera dauma adenovirus (ZdAdV), from a wild bird species, Zoothera dauma (Scaly thrush). This new atadenovirus was discovered by metagenomic sequencing without virus cultivation. Analyses of the full genome sequence revealed that this new virus is a distinct member of the genus Atadenovirus and represents a novel species. ZdAdV has a genome of 34,760 bp with 28 predicted genes and 39% GC content. ZdAdV is the first atadenovirus to contain ORF19, a gene previously found only in aviadenoviruses. Phylogenetic analysis of ORF19 suggests that it was acquired by ZdAdV through horizontal gene transfer from an aviadenovirus. By analyzing all orthologous genes of aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus, we also found potential horizontal gene transfer for the E4 gene in Pigeon aviadenovirus B. Our study widens our knowledge concerning the genetic diversity and evolutionary history of atadenoviruses and their potential for cross-species transmission.

Development of a unique sandwich enzyme-linked immunosorbent assay based on monoclonal antibodies for the specific detection of the egg drop syndrome virus.

RESEARCH HIGHLIGHTS: A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6.The sandwich ELISA maintained high specificity and sensitivity.The sandwich ELISA had equivalent consistency with real-time PCR assay.

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Adenoviruses in Avian Hosts: Recent Discoveries Shed New Light on Adenovirus Diversity and Evolution.

While adenoviruses cause infections in a wide range of vertebrates, members of the genus Atadenovirus, Siadenovirus, and Aviadenovirus predominantly infect avian hosts. Several recent studies on avian adenoviruses have encouraged us to re-visit previously proposed adenovirus evolutionary concepts. Complete genomes and partial DNA polymerase sequences of avian adenoviruses were extracted from NCBI and analysed using various software. Genomic analyses and constructed phylogenetic trees identified the atadenovirus origin from an Australian native passerine bird in contrast to the previously established reptilian origin. In addition, we demonstrated that the theories on higher AT content in atadenoviruses are no longer accurate and cannot be considered as a species demarcation criterion for the genus Atadenovirus. Phylogenetic reconstruction further emphasised the need to reconsider siadenovirus origin, and we recommend extended studies on avian adenoviruses in wild birds to provide finer evolutionary resolution.

Molecular characterization of a bovine adenovirus type 7 (Bovine Atadenovirus F) strain isolated from a systemically infected calf in Germany.

Bovine adenovirus 7 (BAdV-7) is an unclassified member of the genus Atadenovirus with a worldwide distribution and has been reported to induce clinical disease of varying severity in infected cattle, ranging from asymptomatic infections to severe enteric or respiratory disease. In this study, we used next-generation sequencing to obtain the first complete genome sequence of a European strain of BadV-7, from pooled spleen and liver tissue obtained from a deceased newborn Limousin calf. Histopathological analysis and electron microscopy showing systemic lesions in multiple organs with intranuclear amphophilic inclusions observed in endothelial cells in multiple peripheral tissues. Virus isolation was readily achieved from tissue homogenate using bovine esophagus cells (KOP-R), a strategy which should facilitate future in vitro or in vivo BAdV-7 studies. Phylogenetic analysis of available genome sequences of BAdV-7 showed that the newly identified strain groups most closely with a recent BAdV-7 strain, SD18-74, from the USA, confirming that this newly identified strain is a member of the Atadenovirus genus. The fiber gene was found to be highly conserved within BAdV-7 strains but was highly divergent in comparison to Ovine adenovirus 7 (OAdV-7) (39.56% aa sequence identity). Furthermore, we report a variable region of multiple tandem repeats between the coding regions of E4.1 and RH5 genes. In summary, the presented pathological and molecular characterization of this case suggests that further research into the worldwide molecular epidemiology and disease burden of BAdV-7 is warranted.

First Report on Detection and Molecular Characterization of Adenoviruses in the Small Indian Mongoose (Urva auropunctata).

Using a broad-range nested PCR assay targeting the DNA-dependent DNA polymerase (pol) gene, we detected adenoviruses in 17 (20.48%) out of 83 fecal samples from small Indian mongooses (Urva auropunctata) on the Caribbean island of St. Kitts. All 17 PCR amplicons were sequenced for the partial pol gene (~300 bp, hereafter referred to as Mon sequences). Fourteen of the 17 Mon sequences shared maximum homology (98.3-99.6% and 97-98.9% nucleotide (nt) and deduced amino acid (aa) sequence identities, respectively) with that of bovine adenovirus-6 (species Bovine atadenovirus E). Mongoose-associated adenovirus Mon-39 was most closely related (absolute nt and deduced aa identities) to an atadenovirus from a tropical screech owl. Mon-66 shared maximum nt and deduced aa identities of 69% and 71.4% with those of atadenoviruses from a spur-thighed tortoise and a brown anole lizard, respectively. Phylogenetically, Mon-39 and Mon-66 clustered within clades that were predominated by atadenoviruses from reptiles, indicating a reptilian origin of these viruses. Only a single mongoose-associated adenovirus, Mon-34, was related to the genus Mastadenovirus. However, phylogenetically, Mon-34 formed an isolated branch, distinct from other mastadenoviruses. Since the fecal samples were collected from apparently healthy mongooses, we could not determine whether the mongoose-associated adenoviruses infected the host. On the other hand, the phylogenetic clustering patterns of the mongoose-associated atadenoviruses pointed more towards a dietary origin of these viruses. Although the present study was based on partial pol sequences (~90 aa), sequence identities and phylogenetic analysis suggested that Mon-34, Mon-39, and Mon-66 might represent novel adenoviruses. To our knowledge, this is the first report on the detection and molecular characterization of adenoviruses from the mongoose.

Identification and genetic characterization of an isolate of bovine adenovirus 7 from the United States, a putative member of a new species in the genus Atadenovirus.

The bovine adenovirus 7 (BAdV-7) isolate SD18-74 was recovered from lung tissue of calves in South Dakota. The 30,043-nucleotide (nt) genome has the typical organization of Atadenovirus genus members. The sequence shares over 99% nt sequence identity with two Japanese BAdV-7 sequences, followed by 74.9% nt sequence identity with the ovine adenovirus 7 strain OAV287, a member of the species Ovine atadenovirus D. SD18-74 was amplified in both bovine and ovine primary nasal turbinate cells, demonstrating greater fitness in bovine cells. The genomic and biological characteristics of BAdV-7 SD18-74 support the inclusion of the members of the BAdV-7 group in a new species in the genus Atadenovirus.

DETECTION OF DEER ATADENOVIRUS A DNA IN DAM AND OFFSPRING PAIRS OF ROCKY MOUNTAIN MULE DEER (ODOCOILEUS HEMIONUS HEMIONUS) AND ROCKY MOUNTAIN ELK (CERVUS CANADENSIS NELSONI).

Adenovirus hemorrhagic disease affects primarily mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), Rocky Mountain elk (Cervus canadensis nelsoni), and moose (Alces alces) in their first year of life. The method by which the causative virus, Deer atadenovirus A, is maintained in the environment and transmitted to neonates is unknown. In this study, we investigated the potential transmission of the virus from dam to offspring in Rocky Mountain mule deer (Odocoileus hemionus hemionus) and elk in western Wyoming, US. We sampled dams before parturition during placement of vaginal implant transmitters and at parturition and sampled neonates during capture in their first days of life. We also tested for the virus in mortalities submitted for pathologic examination and laboratory analysis. We detected viral DNA in samples from all time points tested but did not find a connection between positive dams and offspring mortalities associated with adenovirus hemorrhagic disease. Although we did not find direct evidence of transmission events between dams and offspring, asymptomatic animals shedding of Deer atadenovirus A, are a likely source of infection in neonates.

Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1.

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine adenovirus 1 (PaAdV-1). The PaAdV-1 genome is 39,664 bp in length, which was the longest atadenovirus to be sequenced, to the best of our knowledge, and contained 42 putative genes. Its genome organisation was characteristic of the members of genus Atadenovirus; however, the novel PaAdV-1 genome was highly divergent and showed the highest sequence similarity with psittacine adenovirus-3 (55.58%). Importantly, PaAdV-1 complete genome was deemed to contain 17 predicted novel genes that were not present in any other adenoviruses sequenced to date, with several of these predicted novel genes encoding proteins that harbour transmembrane helices. Subsequent analysis of the novel PaAdV-1 genome positioned phylogenetically to a distinct sub-clade with all others sequenced atadenoviruses and did not show any obvious close evolutionary relationship. This study concluded that the PaAdV-1 complete genome described here is not closely related to any other adenovirus isolated from avian or other natural host species and that it should be considered a separate species.

FIRST REPORT OF ADENOVIRAL HEMORRHAGIC DISEASE IN THREE MULE DEER (ODOCOILEUS HEMIONUS) IN ARIZONA.

This study presents the gross and histopathological findings of adenoviral hemorrhagic disease (AHD) in two yearling and one adult mule deer (Odocoileus hemionus). These cases represent the first known outbreak of deer adenovirus (Odocoileus adenovirus 1) in Arizona. Over the span of a month, three female captive mule deer were submitted to Midwestern University's Animal Health Institute for postmortem examination. All of these deer were from the same deer farm and historical findings were similar, consisting of acute presentation of hemorrhagic diarrhea and sudden death. Grossly and histopathologically, all cases had severe pulmonary edema and hemorrhagic enteritis. Additionally, two of the three cases had low numbers of large amphophilic intranuclear inclusions expanding endothelial cells within the small intestine and lungs. Viral PCR of pooled small intestine, lung, and spleen from each of the three cases were positive for deer adenovirus and negative for blue tongue and epizootic hemorrhagic disease.

Unravelling the receptor binding property of egg drop syndrome virus (EDSV) from the crystal structure of EDSV fiber head.

Egg drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production, and in the quality of the eggs when it infects chickens. Until now, EDSV virus-cell interactions are poorly understood, and the cellular receptor is still unknown. In the present study, we determined the atomic structure of the fiber head of EDSV (residues 377-644) at 2.74 Šresolution. Structure comparison with the (chick embryo lethal orphan) CELO long fiber head and human adenovirus fiber heads reveals that the avian adenovirus may interact with the same attachment factor in a unique fashion. Based on the previous studies of CELO virus, we assumed that the chicken coxsackievirus and adenovirus receptor (CAR) may be the attachment factor. We then demonstrate that the chicken CAR serves as a cellular attachment factor for EDSV based on three lines of evidences. Taken together, the results presented here are helpful for further exploring the pathogenesis related to the interaction between EDSV and host cells, and may be used for vaccine development and intervention strategies against EDSV infection.

Adenoviruses in free-ranging Australian bearded dragons (Pogona spp.).

Adenoviruses are a relatively common infection of reptiles globally and are most often reported in captive central bearded dragons (Pogona vitticeps). We report the first evidence of adenoviruses in bearded dragons in their native habitat in Australia. Oral-cloacal swabs and blood samples were collected from 48 free-ranging bearded dragons from four study populations: western bearded dragons (P. minor minor) from Western Australia (n = 4), central bearded dragons (P. vitticeps) from central Australia (n = 2) and western New South Wales (NSW) (n = 29), and coastal bearded dragons (P. barbata) from south-east Queensland (n = 13). Samples were tested for the presence of adenoviruses using a broadly reactive (pan-adenovirus) PCR and a PCR specific for agamid adenovirus-1. Agamid adenovirus-1 was detected in swabs from eight of the dragons from western NSW and one of the coastal bearded dragons. Lizard atadenovirus A was detected in one of the dragons from western NSW. Adenoviruses were not detected in any blood sample. All bearded dragons, except one, were apparently healthy and so finding these adenoviruses in these animals is consistent with bearded dragons being natural hosts for these viruses.

IDENTIFICATION OF HELODERMATID ADENOVIRUS 2 IN A CAPTIVE CENTRAL BEARDED DRAGON (POGONA VITTICEPS), WILD GILA MONSTERS (HELODERMA SUSPECTUM), AND A DEATH ADDER (ACANTHOPHIS ANTARCTICUS).

Adenoviruses are medium-sized DNA viruses with very high host fidelity. The phylogenetic relationships of the adenoviruses strongly resemble that of their hosts, consistent with evolutionary codivergence. The genus Atadenovirus appears to have evolved in squamate hosts. Perhaps the best known of the squamate adenoviruses is Agamid adenovirus 1 (AgAdV1), found most commonly in central bearded dragons (Pogona vitticeps), where it is a prevalent cause of hepatitis/enteritis, especially in young animals. All previous reports of adenoviruses in bearded dragons were AgAdV1. Helodermatid adenovirus 2 (HeAdV2) was first seen in Mexican beaded lizards (Heloderma horridus). Subsequently, partial adenoviral polymerase gene sequence from a western bearded dragon (Pogona minor) in Australia was found to share 99% nucleotide homology with HeAdV2. This article reports the discovery of a virus identical to HeAdV2 in a captive central bearded dragon in Florida and wild Gila monsters (Heloderma suspectum) in Arizona. Additionally, a partial adenoviral polymerase gene sharing 98% homology with this HeAdV2 was discovered in a death adder (Acanthophis antarcticus) in Australia. These findings call into question the provenance of HeAdV2. Further studies of atadenoviral host range, diversity of adenoviruses in captive animals, and characterization of adenoviruses from wild squamates are indicated.

RNA-Seq analysis of duck embryo fibroblast cell gene expression during the early stage of egg drop syndrome virus infection.

Egg drop syndrome virus (EDSV), a member of the family Adenoviridae and an economically important pathogen with a broad host range, leads to markedly decreased egg production. However, the molecular mechanism underlying the host-EDSV interaction remains unclear. Here, we performed high-throughput RNA sequencing (RNA-Seq) to study the dynamic changes in host gene expression at 6, 12, and 24 hours post-infection in duck embryo fibroblasts (DEFs) infected with EDSV. Atotal of 441 differentially expressed genes (DEGs) were identified after EDSV infection. Gene Ontology category and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that these DEGs were associated with multiple biological functions, including signal transduction, host immunity, virus infection, cell apoptosis, cell proliferation, and pathogenicity-related and metabolic process signaling pathways. We screened and identified 12 DEGs for further examination by using qRT-PCR. The qRT-PCR and RNA-Seq results were highly consistent. This study analyzed viral infection and host immunity induced by EDSV infection from a novel perspective, and the results provide valuable information regarding the mechanisms underlying host-EDSV interactions, which will prove useful for the future development of antiviral drugs or vaccines for poultry, thus benefiting the entire poultry industry.

Resistance of colostrum-deprived domestic lambs to infection with deer adenovirus.

Seven colostrum-deprived, 3-4-wk-old Rambouillet-Hampshire lambs were inoculated via the mucous membranes with deer adenovirus (DAdV) and monitored for clinical signs for 21 d post-inoculation at which time animals were euthanized and postmortem examinations were performed. Pre-inoculation and post-inoculation serum samples were tested for antibodies to DAdV, ovine adenovirus 7, bovine adenovirus 7, and goat adenovirus 1. Evidence for DAdV infection was determined by virus isolation, PCR tests, and histopathology with immunohistochemistry tests for DAdV. No clinical signs or lesions consistent with adenoviral hemorrhagic disease (AHD) in deer were seen in the lambs, and the lambs did not seroconvert to DAdV. DAdV was not detected by PCR, virus isolation, or immunohistochemistry in any of the samples tested from the lambs. A positive control deer similarly inoculated with DAdV developed fatal AHD 1 wk post-inoculation. Our colostrum-deprived lambs did not become infected when inoculated with DAdV.

Adenoviral hemorrhagic disease in California mule deer, 1990-2014.

We reviewed case records from the California Animal Health and Food Safety (CAHFS) laboratory and the California Department of Fish and Wildlife (CDFW) spanning 25 years (1990-2014) for all deer accessions submitted to CAHFS for pathology and/or histopathology, with and without a diagnosis of adenoviral hemorrhagic disease (AHD), in order to determine the prevalence of AHD in California. We also examined spatial and temporal distribution, age, and mule deer subspecies in deer that died from AHD. Of 483 deer submitted to CAHFS for diagnostic testing in 1990-2014, 17.2% were diagnosed with confirmed AHD, and 26.5% were confirmed plus suspected cases of AHD. Columbian black-tailed deer ( Odocoileus hemionus columbianus), particularly fawns and juveniles, were most frequently affected. Deer adenovirus ( Odocoileus adenovirus 1; OdAdV-1) was detected by immunohistochemistry in archived CDFW formalin-fixed, paraffin-embedded tissues from deer that died in mortality events in 1981, 1983, and 1986-1987. OdAdV-1 is a common cause of hemorrhagic disease mortality events in California deer, and mortality as a result of AHD is documented as early as 1981.

Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus.

BACKGROUND: Egg drop syndrome (EDS), caused by the adenovirus "egg drop syndrome virus" (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV. RESULTS: For our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40-45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses. CONCLUSIONS: The direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

Structure of a Reptilian Adenovirus Reveals a Phage Tailspike Fold Stabilizing a Vertebrate Virus Capsid.

Although non-human adenoviruses (AdVs) might offer solutions to problems posed by human AdVs as therapeutic vectors, little is known about their basic biology. In particular, there are no structural studies on the complete virion of any AdV with a non-mammalian host. We combine mass spectrometry, cryo-electron microscopy, and protein crystallography to characterize the composition and structure of a snake AdV (SnAdV-1, Atadenovirus genus). SnAdV-1 particles contain the genus-specific proteins LH3, p32k, and LH2, a previously unrecognized structural component. Remarkably, the cementing protein LH3 has a trimeric ß helix fold typical of bacteriophage host attachment proteins. The organization of minor coat proteins differs from that in human AdVs, correlating with higher thermostability in SnAdV-1. These findings add a new piece to the intriguing puzzle of virus evolution, hint at the use of cell entry pathways different from those in human AdVs, and will help development of new, thermostable SnAdV-1-based vectors.

Whole-genome sequences of Odocoileus hemionus deer adenovirus isolates from deer, moose and elk are highly conserved and support a new species in the genus Atadenovirus.

We present the first complete genome sequence of Odocoileus hemionus deer adenovirus 1 (OdAdV-1). This virus can cause sporadic haemorrhagic disease in cervids, although epizootics with high mortality have occurred in California. OdAdV-1 has been placed in the genus Atadenovirus, based on partial hexon, pVIII and fibre genes. Ten field isolates recovered from naturally infected mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginiana) and moose (Alces alces) from Wyoming, black-tailed deer (Odocoileus hemionus columbianus) from California, and Rocky Mountain elk (Cervus elaphus nelsoni) from Colorado and Washington state were sequenced. The genome lengths ranged from 30 620 to 30 699 bp, contained the predicted proteins and gene organization typical of members of genus Atadenovirus, and had a high percentage of A/T nucleotides (66.7 %). Phylogenic analysis found that the closest ancestry was with ruminant atadenoviruses, while a divergence of the hexon, polymerase and penton base proteins of more than 15 % supports classification as a new species. Genetic global comparison between the 10 isolates found an overall 99 % identity, but greater divergence was found between those recovered from moose and elk as compared to deer, and a single variable region contained most of these differences. Our findings demonstrate that OdAdV-1 is highly conserved between 10 isolates recovered from multiple related cervid species, but genotypic differences, largely localized to a variable region, define two strains. We propose that the virus type name be changed to cervid adenovirus 1, with the species name Cervid atadenovirus A. Sequence data were used to develop molecular assays for improved detection and genotyping.