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Identification of aviadenovirus and dependoparvovirus in an Adélie penguin fecal sample from Cape Royds (Ross Island, Antarctica).

Adenoviruses (Adenoviridae) and their associated replication-deficient dependoparvoviruses (Parvoviridae) are recognized as significant components of the avian virome. While well-studied in domestic birds, these viruses remain underexplored in wild Antarctic birds. Information is limited on viruses circulating in penguins, particularly outside of the Antarctic Peninsula Region. Here we leverage a fecal sample collected for diet analyses to identify an aviadenovirus (37,731 nt) and two distinct dependoparvoviruses. The Ad&#xe9;lie penguin (Pygoscelis adeliae) feces-derived adenovirus exhibits a genome organization consistent with members of the genus Aviadenovirus but shares only ~64% amino acid identity in the DNA polymerase protein with its closest known relative and thus represents a new species of aviadenovirus identified in penguins. Additionally, we identified two new dependoparvoviruses, one in length of 4,869 nt and a second of 4,162 nt. These two dependoparvoviruses are diverse, sharing <61% Rep amino acid identity with those of other dependoparvoviruses. Penguin dependoparvovirus 1 represents a new species. The co-detection of these viruses in a single seasonal sample could suggest a potential helper-satellite relationship in Ad&#xe9;lie penguins. This study provides the first evidence of aviadenoviruses in Ad&#xe9;lie penguin feces. These findings highlight the importance of surveillance to understand pathogen dynamics in isolated Antarctic populations.

Animals↗

The complete nucleotide sequence of the egg drop syndrome virus: an intermediate between mastadenoviruses and aviadenoviruses.

The complete nucleotide sequence of an avian adenovirus, the egg drop syndrome (EDS) virus, was determined. The total genome length is 33,213 nucleotides, resulting in a molecular weight of 21.9 x 10(6). The GC content is only 42.5%. Between map units 3.5 and 76.9, the distribution of open reading frames with homology to known genes is similar to that reported for other mammalian and avian adenoviruses. However, no homologies to adenovirus genes such as E1A, pIX, pV, and E3 could be found. Outside this region, several open reading frames were identified without any obvious homology to known adenovirus proteins. In the region organized similarly as other adenoviral genomes, most homologies were found to an ovine adenovirus (OAV strain 287). The highest level of amino acid identity was found for the hexon proteins of EDS and OAV. The virus-associated RNA (VA RNA) was identified thanks to the homology with the VA RNA of fowl adenovirus serotype 1 (FAV1). Similarities with FAV1 were also found in the fiber protein. Our results demonstrate that the avian EDS virus represents an intermediate between mammalian and avian adenoviruses. The nucleotide sequence and genomic organization of the EDS virus reflect the heterogeneity of the aviadenovirus genus and the Adenoviridae family.

Adenoviridae↗

Characterization of the haemorrhagic enteritis virus genome and the sequence of the putative penton base and core protein genes.

Haemorrhagic enteritis virus (HEV) is a member of a genetically ill-defined group within the genus Aviadenovirus which causes significant clinical disease in gallinaceous fowl. Using DNA obtained from a low virulence isolate of HEV passed in turkeys, we developed a genomic restriction map and estimated an apparent genomic length of 25.5 kb. No evidence for extensive DNA hybridization was found between the HEV genome and either the hexon or penton base genes of human adenovirus 2 (HAdV-2) and fowl adenovirus 10 (FAdV-10). The HEV penton base gene was identified by PCR using primers based on conserved adenoviral DNA sequences. The penton base gene was expressed in Escherichia coli as a fusion protein and detected by anti-HEV serum in both colony and denaturing gel immunoblots. DNA sequencing revealed a putative penton base ORF with a predicted amino acid sequence showing approximately 39.0%, 53.0% and 44.2% similarity with the penton base of HAdV-2, human adenovirus 40 (HAdV-40) and FAdV-10, respectively. The penton base gene was located at 43.3-48.6 m.u. on the HEV genome and had a remarkably low G+C content (33.8%). DNA sequencing also revealed ORFs for putative core proteins resembling pVII, p-mu and a partial ORF similar to pVI (hexon-associated protein) of HAdV-2 and HAdV-40. The results support the claim that HEV represents a distinct group of viruses within the genus Aviadenovirus.

Amino Acid Sequence↗

Diagnosis of avian adenovirus infections using DNA in situ hybridization.

Three DNA oligonucleotide probes designated FN-23, FN-48, and FN-96 were evaluated for the diagnosis of aviadenovirus infections by DNA in situ hybridization. Paraffin-embedded tissues were obtained from birds with confirmed adenovirus infection, birds with putative adenovirus infections, and birds with intranuclear inclusions caused by herpesvirus and polyomavirus. In birds with confirmed adenovirus infection, probes FN-23 and FN-96 identified 78% and 72% of diseased individuals, respectively. Only probe FN-48 detected chickens with group II adenovirus infection. In birds with putative adenovirus infection, the DNA probes confirmed aviadenovirus infections in 76% of the population. Probes FN-23, FN-96, and FN-48 detected 85%, 74%, and 18% of adenovirus-infected birds, respectively. None of the DNA probes cross-hybridized with tissues from polyomavirus-infected psittaciform birds or with tissues from a chicken with infectious laryngotracheitis. In contrast, probe FN-23 did cross-hybridize to herpesvirus-infected tissues from two of eight psittaciform birds with Pacheco's parrot disease. Probes FN-48 and FN-96 did not react with these tissues.

Adenoviridae Infections↗

An ovine adenovirus vector lacks transforming ability in cells that are transformed by AD5 E1A/B sequences.

Adenoviruses of the Mastadenovirus and Aviadenovirus genera are able to transform certain cell types and induce tumor formation in susceptible animals. For the mastadenoviruses the E1A/B sequences are largely responsible for these properties but E4 sequences may also be involved. The transforming sequences of the aviadenoviruses, which lack E1A/B and E4 homologues, have not yet been fully identified. The recent proposal for a third genus of adenoviruses, which apparently lack an E1A homologue and have weak E1B homology, prompted an examination of the transforming properties of ovine adenovirus OAV287 (OAV), the prototype member of the new group. When OAV and human adenovirus type 5 (Ad5) were used to infect primary rat embryo cells, transformed foci developed in Ad5- but not in OAV-infected cultures. Similarly, after plasmid transfection, baby rat kidney cells were transformed by Ad5 E1A/B but not by OAV sequences. When CSL503 cells, an ovine cell line that is permissive for OAV, were transfected with Ad5 E1A/B sequences, transformed foci again appeared. However, plasmids or fragments containing complete or partial OAV genome sequences did not detectably transform CSL503 cells under the same conditions. When Ad5 E1A/B sequences were incorporated into the complete OAV genome and transfected, transformed clones were again obtained, showing that the gene dosage and transfection conditions were not limiting for transformation. The provision of Ad5 E1A and OAV sequences in combination marginally increased the number of morphologically altered foci in baby rat kidney cells but failed to induce multilayered focus formation. The data suggest that OAV lacks transforming functions in the cell types examined. Additional information suggesting that OAV may have a fundamentally distinct strategy for replication compared with other Ads is discussed.

Adenoviridae↗

The hydropericardium syndrome and inclusion body hepatitis in domestic fowl.

Hydropericardium syndrome, an emerging disease of poultry, has recently been detected in some countries of Asia and America, particularly in broiler birds aged 3-6 weeks. The disease is characterized by its sudden occurrence with high mortality of up to 80% in broilers and low mortality of under 10% in layers, associated with hydropericardium. Its course is of 7-15 days under natural conditions. The causative agent is probably fowl adenovirus serotype 4, belonging to group I aviadenovirus genus of the family adenoviridae, which can be cultivated in primary cell cultures of chicken kidney and embryo liver cells. The transmission of disease occurs laterally by the oral-faecal route. The livers of affected birds show necrotic foci, and basophilic intranuclear inclusion bodies fill the entire enlarged nucleus of some of the hepatocytes. The disease can be diagnosed from its gross lesions, histopathological changes in the liver and by serological tests, such as agar gel diffusion, counter immunoelectrophoresis, indirect haemagglutination and ELISA. It has been brought under control by inactivated liver organ vaccines (0.25 ml/bird) or inactivated cell culture vaccines (10(3.5) LD50/bird) given by the subcutaneous route at 10-15 days of age. The vaccine is effective in the face of an outbreak and significantly reduces the mortality.

Adenoviridae Infections↗

DNA sequence of frog adenovirus.

The genome of frog adenovirus (FrAdV-1) was sequenced and found to be the smallest of all known adenovirus genomes. The sequence obtained was 26163 bp in size and contains a substantial direct repeat near the right terminus, implying that it was derived by recombination from a parental genome of only 25517 bp. The closest relative of FrAdV-1 proved to be turkey adenovirus 3, an avian adenovirus with no previously known near relative. Sequence comparisons showed that the two viruses have equivalent gene complements, including one gene the product of which is related to sialidases. Phylogenetic analyses supported the establishment of a fourth adenovirus genus containing these two viruses, in addition to the established genera Mastadenovirus: and Aviadenovirus: and the proposed genus Atadenovirus: Sixteen genes were identified as being conserved between these four lineages and were presumably inherited from an ancestral adenovirus.

Adenoviridae↗

Characterization of a new species of adenovirus in falcons.

In 1996, a disease outbreak occurred at a captive breeding facility in Idaho, causing anorexia, dehydration, and diarrhea or sudden death in 72 of 110 Northern aplomado falcons (Falco femoralis septentrionalis) from 9 to 35 days of age and in 6 of 102 peregrine falcons (Falco peregrinus) from 14 to 25 days of age. Sixty-two Northern aplomado and six peregrine falcons died. Epidemiologic analyses indicated a point source epizootic, horizontal transmission, and increased relative risk associated with cross-species brooding of eggs. Primary lesions in affected birds were inclusion body hepatitis, splenomegaly, and enteritis. The etiology in all mortalities was determined by molecular analyses to be a new species of adenovirus distantly related to the group I avian viruses, serotypes 1 and 4, Aviadenovirus. In situ hybridization and PCR demonstrated that the virus was epitheliotropic and lymphotropic and that infection was systemic in the majority of animals. Adeno-associated virus was also detected by PCR in most affected falcons, but no other infectious agents or predisposing factors were found in any birds. Subsequent to the 1996 epizootic, a similar disease caused by the same adenovirus was found over a 5-year period in orange-breasted falcons (Falco deiroleucus), teita falcons (Falco fasciinucha), a merlin (Falco columbarius), a Vanuatu peregrine falcon (Falco peregrinus nesiotes), and gyrfalcon x peregrine falcon hybrids (Falco rusticolus/peregrinus) that died in Wyoming, Oklahoma, Minnesota, and California. These findings indicate that this newly recognized adenovirus is widespread in western and midwestern North America and can be a primary pathogen in different falcon species.

Adenoviridae Infections↗

Isolation and epidemiology of falcon adenovirus.

An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2005). In this study, the virus was successfully isolated and propagated in peregrine falcon embryo fibroblasts, in which it caused visible and reproducible cytopathology. Testing for serum neutralizing antibodies found that infection with this virus was limited almost exclusively to falcons. Serology also found that wild and captive peregrine falcons had high seropositivity rates of 80% and 100%, respectively, although clinical disease was rarely reported in this species. These data implicate peregrine falcons as the natural host and primary reservoir for the virus. Other species of North American falcons, including aplomado falcons, had lower seropositivity rates of 43 to 57%. Falcon species of tropical and/or island origin were uniformly seronegative, although deaths among adults of these species have been described, suggesting they are highly susceptible. Chickens and quail were uniformly seronegative and not susceptible to infection, indicating that fowl were not the source of infection. Based on the information from this study, the primary control of falcon adenovirus infections should be based on segregation of carrier and susceptible falcon species.

Adenoviridae Infections↗

Falcon adenovirus infection in breeding Taita falcons (Falco fasciinucha).

Four female and 3 male Taita falcons (Falco fasciinucha) out of a breeding colony of 14 Taita falcons (7 pairs) died during the breeding season after showing lethargy and anorexia for 1 to 2 days. All animals were submitted for necropsy. Gross lesions in the female falcons were characterized by anemia secondary to marked hemorrhage into the ovary and oviduct, serofibrinous effusion into the cardioabdominal cavity and serosal petechiae. In addition, marked necrotizing splenitis and pulmonary hemorrhage were present. Histologically, the female falcons had mild necrotizing hepatitis with numerous intranuclear inclusion bodies and necrotizing splenitis with rare inclusion bodies. There were no gross lesions in the male falcons, and the histological lesions were characterized by urate deposition and rare intranuclear inclusion bodies in the renal tubular epithelial cells. Adenoviral particles were found by electron microscopy in the cloacal contents of the female Taita falcons but not in the male falcons. DNA in situ hybridization revealed widespread aviadenoviral nucleic acid within the nuclei of hepatocytes, renal tubular epithelial cells, and adrenal cells in the female falcons but no aviadenoviral nucleic acid in 1 male falcon and only a low quantity of adenoviral nucleic acid in the liver and kidney of another male Taita falcon. PCR amplified aviadenoviral DNA in the liver and intestine of all Taita falcons. The amplicons were sequenced, and the virus was identified as falcon adenovirus. The deaths of the female and male birds were attributed to the aviadenovirus infection.

Adenoviridae Infections↗

Protection of turkeys from hemorrhagic enteritis with a recombinant fowl poxvirus expressing the native hexon of hemorrhagic enteritis virus.

Hemorrhagic enteritis (HE) is an economically important disease of turkeys caused by a type II aviadenovirus, hemorrhagic enteritis virus (HEV). The vaccines currently available to the commercial poultry producer are highly effective in preventing disease outbreaks; however, they are immunosuppressive. A recombinant fowl poxvirus (rFPV) expressing the native hexon of HEV has been shown to induce an anti-HEV humoral immune response in turkeys. In this study, the rFPV expressing the native hexon of HEV was compared with a commercial HEV vaccine (vxHEV) for its ability to protect turkeys from virulent HEV challenge. Complete protection from the enteritis of HE was achieved in experimental groups vaccinated with either the rFPV or the vxHEV. Lymphocyte stimulation was measured in turkeys inoculated with rFPV, vxHEV, or a sublethal dose of HEV or not inoculated. No statistically significant immunodepression was observed in turkeys receiving the rFPV.

Adenoviridae Infections↗

Isolation of etiological agent of hydropericardium syndrome in chicken embryo liver cell culture and its serological characterization.

The virus causing hydropericardium syndrome was isolated in chicken embryo liver (CEL) cell culture from livers obtained from naturally infected broilers. The cytopathic effects characterized by rounding and degeneration of cells were visible 36 hr post infection in first passage. At 4th passage level, the infectivity titre was 5.24 log10 TCID50/ml. In May-Grunwald and Giemsa stained cells, basophilic intranuclear inclusions ('bird eye' inclusion), typical of aviadenovirus infection, were observed. The specificity of inclusion was confirmed by indirect immunofluorescence. Various serological tests, such as agar gel precipitation test, counter immuno electrophoresis, micro serum neutralization test and enzyme linked immunosorbent assay were also standardized to confirm the isolation of etiological agent of hydropericardium syndrome in CEL cell culture and to diagnose the disease in poultry.

Adenoviridae Infections↗

Close phylogenetic relationship between egg drop syndrome virus, bovine adenovirus serotype 7, and ovine adenovirus strain 287.

A cloned egg drop syndrome (EDS) virus genomic DNA fragment containing the protease gene has been identified and the complete nucleotide sequence of the protease and partial nucleotide sequence of the hexon genes has been determined. Phylogenetic analysis of the protease gene has revealed EDS virus to be genetically more closely related to bovine adenovirus type 7 (BAV-7) and ovine adenovirus isolate 287 (OAV287) than either of these two viruses are to other members of the genus Mastadenovirus or EDS virus is to an other member of the Aviadenovirus genus. The three viruses share further similarities in that they have a high percentage AT content in their genome and are characterized by having more compact genomes than other adenoviruses. The protease gene from all three viruses contained the active site residues (H55-D72-C122 triad) and C104 (providing a disulfide bond to cofactor pVIc). However, P137, found in all other members of the Mastadenovirus genus, and thought to be involved in trafficking, was missing from the protease of the EDS virus, BAV-7, and OAV287. These results suggest that EDS virus should be classified along with BAV-7 and OAV287 in a separate taxon.

Adenoviridae↗

Gene expression by atypical recombinant ovine adenovirus vectors during abortive infection of human and animal cells in vitro.

An bovine adenovirus, which is phylogenetically distinct from the Mastadeno- and Aviadenoviruses, was used to construct recombinants in which reporter genes were expressed from the OAV major late, or human cytomegalovirus promoters. It was demonstrated by transgene expression that OAV could infect bovine nasal turbinate and rabbit kidney cells as well as a range of human cell types, including lung and foreskin fibroblasts as well as liver, prostate, breast, colon, and retinal lines. Some human lines, e.g., 293 and LNCaP were not detectably infected. Infection occurred even though OAV has a fiber protein with a unique cell binding domain and a penton protein that lacks the integrin-binding Arg-Gly-Asp motif which facilitates entry by human adenoviruses. Most cell lines showed little or no ill effect for several days after infection but a prominent cytopathic effect appeared in fibroblasts after 3-4 days. However, no viral DNA synthesis was detected and replication was abortive. Viral promoter activity during infection of nonpermissive cell types was assayed by RT-PCR. Early promoter activity was detectable in some, but not all cell types. In a liver and a colon carcinoma cell line, none of the promoters examined was significantly active, even when a higher multiplicity of infection was used. Major late promoter activity was not detectable in any cell type. The lack of DNA replication and MLP function suggests that a critical transition from early to late gene expression does not occur during abortive infection by OAV.

Adenoviridae↗

Identification of transcripts and promoter regions of ovine adenovirus OAV287.

The ovine adenovirus isolated OAV287 represents a new group of adenoviruses that are distinct from the Mast- and Aviadenoviruses by several criteria, including genome arrangement. The OAV major late promoter and some late transcripts were previously mapped. To better define the probable coding sequences and to identify the approximate location of early promoters a partial transcription map of the genome was elucidated using a PCR-based approach. This was possible because the complete nucleotide sequence of the genome was known. The strategy permitted the identification of transcription start sites and RNA splice junctions and allowed the approximate location of promoters in the lefthand end, IVa2, E2, P32K, and E4 regions to be deduced. The data showed that lefthand end and E4 regions are controlled by three and two temporally distinct promoters, respectively. The E2 region is controlled by a single promoter, in contrast to Mastadenoviruses, where E2 expression is controlled by the E2A and E2B promoters. The p32kDa structural protein at the lefthand end and the IVa2 protein are also expressed from their own promoters. These data contribute to the first overview of transcription from a non-Mastadenovirus genome.

Amino Acid Sequence↗

A proposal for a new (third) genus within the family Adenoviridae.

This article presents a proposal for the establishment of a new adenovirus genus to accommodate certain bovine, ovine, and avian adenoviruses with special characteristics which differentiate them from members of the existing genera Mastadenovirus and Aviadenovirus. This proposal has been developed from earlier versions with advice from the Adenovirus Study Group of the International Committee on Taxonomy of Viruses (ICTV).

Adenoviridae↗

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1.

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth.

Adenoviridae↗

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes.

Most adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 (Aviadenovirus), DAdV-1, and BAdV-4 (Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy.

Adenoviridae↗