Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Novel adenoviruses from captive psittacine birds in Slovenia.

To assess the prevalence of adenoviruses in psittacine birds kept in Slovenia, 258 cloacal swabs were collected from different psittacine species and screened by a nested PCR with degenerate, consensus primers targeting the adenoviral DNA polymerase gene. Forty-two samples were found to be positive. By sequencing, 28 samples from 10 different parrot species were identified as the formerly described siadenovirus, psittacine adenovirus 2 (PsAdV-2). A second siadenovirus, a variant of PsAdV-5 (described earlier from Pacific parrotlet, sun parakeet, cockatiel and budgerigar) was found in seven budgerigars, two cockatiels and an amazon parrot species. A variant of Meyer's parrot adenovirus (aviadenovirus, proposed PsAdV-8) was identified in an African grey parrot and a cockatiel. Two novel atadenoviruses were revealed in cockatiel (PsAdV-9) and rose-ringed parakeet (PsAdV-10). These results support the earlier finding that many PsAdVs can cross the species barrier among psittacines, especially effectively in the case of PsAdV-2.

Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Human species D adenovirus hexon capsid protein mediates cell entry through a direct interaction with CD46.

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.

Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 56.

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

Detection and partial genetic characterisation of novel avi- and siadenoviruses in racing and fancy pigeons (Columba livia domestica).

Up to now, only a single adenovirus (AdV) isolate seemingly specific for pigeons, hence named pigeon AdV-1 (PiAdV-1), has been characterised at DNA sequence level. In the present work, the prevalence and diversity of AdVs occurring in domestic pigeon were examined by a survey performed on randomly collected samples using a very efficient, consensus nested PCR targeting the viral DNA polymerase gene. The newly detected viruses were characterised by sequencing and phylogeny analysis. Amplification of additional genome fragments was attempted by the use of several other PCR methods aiming at the hexon gene. During a 4-year survey, samples from dead or live, healthy pigeons originating from 27 lofts were examined in Hungary. Almost 50% of the samples (48 out of 97) proved to be positive for AdV. Sequence analysis revealed the presence of four hitherto unknown pigeon AdV types. PiAdV-1 was also identified in one sample. Two novel viruses named PiAdV-2 and -3 were found to belong to the genus Aviadenovirus, and two other novel types (PiAdV-4 and -5) to the genus Siadenovirus. This is the first report on the occurrence of siadenoviruses in birds belonging to the order Columbiformes. Approximately two-thirds of the PiAdV-2 genome was sequenced and analysed.

Crystal structure of raptor adenovirus 1 fibre head and role of the beta-hairpin in siadenovirus fibre head domains.

BACKGROUND: Most adenoviruses recognize their host cells via an interaction of their fibre head domains with a primary receptor. The structural framework of adenovirus fibre heads is conserved between the different adenovirus genera for which crystal structures have been determined (Mastadenovirus, Aviadenovirus, Atadenovirus and Siadenovirus), but genus-specific differences have also been observed. The only known siadenovirus fibre head structure, that of turkey adenovirus 3 (TAdV-3), revealed a twisted beta-sandwich resembling the reovirus fibre head architecture more than that of other adenovirus fibre heads, plus a unique beta-hairpin embracing a neighbouring monomer. The TAdV-3 fibre head was shown to bind sialyllactose. METHODS: Raptor adenovirus 1 (RAdV-1) fibre head was expressed, crystallized and its structure was solved and refined at 1.5 Å resolution. The structure could be solved by molecular replacement using the TAdV-3 fibre head structure as a search model, despite them sharing a sequence identity of only 19 %. Versions of both the RAdV-1 and TAdV-3 fibre heads with their beta-hairpin arm deleted were prepared and their stabilities were compared with the non-mutated proteins by a thermal unfolding assay. RESULTS: The structure of the RAdV-1 fibre head contains the same twisted ABCJ-GHID beta-sandwich and beta-hairpin arm as the TAdV-3 fibre head. However, while the predicted electro-potential surface charge of the TAdV-3 fibre head is mainly positive, the RAdV-1 fibre head shows positively and negatively charged patches and does not appear to bind sialyllactose. Deletion of the beta-hairpin arm does not affect the structure of the raptor adenovirus 1 fibre head and only affects the stability of the RAdV-1 and TAdV-3 fibre heads slightly. CONCLUSIONS: The high-resolution structure of RAdV-1 fibre head is the second known structure of a siadenovirus fibre head domain. The structure shows that the siadenovirus fibre head structure is conserved, but differences in the predicted surface charge suggest that RAdV-1 uses a different natural receptor for cell attachment than TAdV-3. Deletion of the beta-hairpin arm shows little impact on the structure and stability of the siadenovirus fibre heads.

Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses.

UNLABELLED: E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCE: Two early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of the Human mastadenovirus A, F, and G species. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3.

The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus.

BACKGROUND: In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus. METHODS: We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414-535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution. RESULTS: Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed. CONCLUSIONS: Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.

A novel siadenovirus detected in the kidneys and liver of Gouldian finches (Erythura gouldiae).

A novel siadenovirus was found in six captive Gouldian finches (Erythrura gouldiae) in the United States and Hungary. Histopathological examination revealed inclusions in the kidney of the captive Gouldian finch in the United States, and virions morphologically consistent with adenoviruses were seen by electron microscopy. Partial sequence of the DNA-dependent DNA polymerase gene was gained by consensus PCR and sequencing in all six finches, and all proved to be identical. In one Hungarian finch, additional sequence was obtained from the DNA polymerase gene, the pre-terminal protein (pTP) gene, the 52k gene, and the hexon gene. Bayesian, maximum likelihood, and distance-based analyses showed the novel virus clusters with the siadenoviruses, and is herein referred to as Gouldian finch adenovirus 1. The genes looked at in this study had low G+C percentages, which is common in the genus Siadenovirus, and suggestive of recent host switch. The significance of this virus' presence is unknown at this time as clinical signs of positive birds varied.

Whole-genome sequences of two turkey adenovirus types reveal the existence of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus.

There are eight species established for aviadenoviruses: Fowl adenovirus A-E, Goose adenovirus A, Falcon adenovirus A and Turkey adenovirus B. The aim of this study was to sequence and analyse the complete genomes of turkey adenovirus 4 (TAdV-4) and TAdV-5 (strain 1277BT) in addition to almost two-thirds of the genome of another TAdV-5 strain (strain D1648). By applying next-generation sequencing, the full genomes were found to be 42 940 and 43 686 bp and the G+C content was 48.5 and 51.6 mol% for TAdV-4 and TAdV-5, respectively. One fiber gene was identified in TAdV-4, whereas two fiber genes were found in TAdV-5. The genome organization of TAdV-4 resembled that of fowl adenovirus 5 (FAdV-5), but it had ORF1C near the left end of the genome. TAdV-4 also had five 123 bp tandem repeats followed by five 33 bp tandem repeats, but they occurred before and not after ORF8, as in several fowl adenoviruses. The genome organization of TAdV-5 was almost the same as that of FAdV-1 but with a possible difference in the splicing pattern of ORF11 and ORF26. Phylogenetic analyses and G+C content showed differences that seem to merit the establishment of two new species within the genus Aviadenovirus: Turkey adenovirus C (for TAdV-4) and Turkey adenovirus D (for TAdV-5). Our analyses suggest a common evolutionary origin of TAdV-5 and FAdV-1.

Crystallization of the C-terminal head domain of the fibre protein from a siadenovirus, turkey adenovirus 3.

Turkey adenovirus 3 belongs to the genus Siadenovirus. Its predicted fibre protein consists of an N-terminal virus-attachment domain, a central shaft domain and a head domain at the C-terminus. The head domain has little sequence identity to known adenovirus fibre head structures. Crystals of the fibre head domain consisting of amino acids 304-454 with an N-terminal purification tag were produced. Crystals of native and selenomethionine-derivatized protein belonged to space group I23 (unit-cell parameter 99 Å). They diffracted synchrotron radiation to 2.0 and 2.14 Šresolution, respectively, and are expected to contain one monomer in the asymmetric unit.