Otus scops adenovirus: the complete genome sequence of a novel aviadenovirus discovered in a wild owl

We present the complete genome sequence of an aviadenovirus obtained by metagenomics from cloacal swabs taken from a free-living Eurasian scops owl (Otus scops, a small raptor distributed in Europe and several parts of Asia) in China. Thirty protein coding genes were predicted in this 40,239-bp-long genome, which encodes the largest fiber protein among all reported aviadenoviruses. The viral genome sequence is highly divergent, and the encoded proteins have an average of only 55% amino acid sequence identity to those of other adenoviruses. In phylogenetic analysis, the new owl virus grouped with members of the genus Aviadenovirus and formed a common clade with another owl adenovirus reported previously in Japan. This is the second complete genome sequence of an aviadenovirus discovered in owls, and its proteins have an average of 62% amino acid sequence identity to those of the previously reported owl adenovirus. Combining this result with comparative genomic analysis of all aviadenoviruses, we propose that this owl virus and the previously described Japanese owl adenovirus can be assigned to two new species in the genus Aviadenovirus. This study provides new data on the diversity of aviadenoviruses in wild birds.

infect birds and can cause infectious diseases in many cases. In the early stage of adenovirus research, discoveries of new adenoviruses, such as fowl aviadenovirus A, were mainly reported in poultry. Clinical signs of adenovirus infection in domestic fowl may include inclusion body hepatitis, egg drop syndrome, hemorrhagic enteritis, gizzard erosion, and hydropericardium syndrome [3][4][5][6][7][8]. With the development of new research methods, especially the advent of highthroughput sequencing, more adenoviruses have been discovered in hosts other than poultry [9]. So far, NCBI has recorded 194 genomic assemblies of members of more than 15 aviadenovirus species. Many of these genome sequences were obtained from wild birds. As the main reservoirs for zoonotic viruses, wild animals show high potential and importance for discovery of unknown viruses [10]. In recent years, studies on wild birds have shown that the diversity of adenoviruses in wildlife may far exceed that which is currently known [11][12][13].
The scops owl belongs to the family Strigidae within the order Strigiformes. It is a relatively small nocturnal predator that lives in Europe and parts of Asia, often in very close proximity to human settlements. Wild birds are the natural hosts of adenoviruses, including atadenoviruses, siadenoviruses, and aviadenoviruses. There have been only a few reports about adenoviruses in free-living owls. The first report that owls can harbor an adenovirus was in 2004. Zsivanovits et al. reported an outbreak of an adenoviral disease that occurred in a raptor collection in the United Kingdom [14]. In that case, a 1-year-old Bengal eagle owl (Bubo bengalensis) and a 3-year-old Verreaux's eagle owl (Bubo lacteus) died due to an infection with a novel siadenovirus, which was then sequenced (NCBI accession no. EU715130) and designated as raptor adenovirus 1 (RAdV-1) [15]. In 2016, Komatsu et al. reported that tropical screech owl (Megascops choliba) chicks in one domestic breeding facility died with typical signs of adenovirus infection (referred to here as OlAdV) [16]. This adenovirus was identified as an aviadenovirus by PCR and sequencing the DNA polymerase gene, and it was the first and only aviadenovirus found in owls. The complete genome of an identical aviadenovirus (NCBI accession number: LC638697), isolated at the same breeding facility from dead chicks of six other owl species, was sequenced and analyzed [17]. Thus, we were able to perform a detailed comparative genomic analysis of the Japanese isolate (OlAdV) and our newly discovered scops owl adenovirus (OsAdV). In the present study, we collected swab samples from the cloaca of Otus scops, and a novel aviadenovirus, which we have named "Otus scops adenovirus" (OsAdV) was identified by metagenomic sequencing. The complete genome sequence of this novel virus was assembled, annotated, and analyzed.
Eight scops owls (Otus scops) were captured with nets on an island off the east coast of China (37.9686°N, 120.6144°E) in September 2018. Wild birds were collected, sampled without any biological perturbation, and immediately released. Cloacal swabs were pooled, DNA was extracted, and the OsAdV genome sequence was obtained by metagenomic sequencing. The full genome of OsAdV is 40,239 bp in size and has a G+C content of 48%. It was found to contain 30 ORFs. The deduced amino acid (aa) sequences of all of the viral proteins were aligned with those in the NR (NCBI RefSeq non-redundant proteins) and SWISS-PROT databases. Based on these alignments, 29 of the 30 genes were functionally annotated. The first ORF at the left end of the genome did not match any genes of known function. The positions and coding directions of all genes are shown in the schematic genome plot (Fig. 1). ORF1, ORF2, 52-kDa protein, pIIIa, pIII, pVII, pX, pVI, hexon, protease, 100-kDa protein, 22-kDa protein, 33-kDa protein, pVIII, fiber, and GAM-1 genes were identified on the rightward transcribed (r) genomic strand. On the complementary (l) strand, in the opposite orientation, ORF14A, ORF14, ORF13, ORF12, IVa2, DNA polymerase, pTP, DBP, ORF22, ORF20, ORF19, and ORF19A were found. The complete nucleotide sequence and gene annotation of OsAdV were deposited in the GenBank database (accession number ON843719). The dataset of paired-end raw reads was submitted to NCBI under accession no. SRR19372317.
The DNA polymerase genes of OsAdV and 21 adenoviruses with complete genome sequences available were selected for phylogenetic analysis. Murine mastadenovirus A was chosen as an outgroup to root the tree. Phylogenetic analysis based on the DNA polymerase gene indicated that OsAdV belongs to the genus Aviadenovirus in the family Adenoviridae (Fig. 2). A comparison with other aviadenoviruses showed that OsAdV formed a common clade with the owl adenovirus (OlAdV) found in Japan, which clustered closest to falcon aviadenovirus A. The aa sequence identity in the DNA polymerases of these two viruses is 80%, which means that the divergence is higher than the 15% used to distinguish aviadenoviruses species. Based on these results, we conclude that OsAdV represents a new adenovirus species in the genus Aviadenovirus. We propose that two new species should be established in the genus Aviadenovirus for these owl adenoviruses.
Ortholog analysis was performed for all proteins encoded by OsAdV and 20 typical aviadenoviruses with complete genome sequences (with protein annotation) using OrthoFinder. These genome sequences represent 20 serotypes of 15 species. The protein sequences are based on public records in the NCBI database. When compared to fowl aviadenoviruses, several putative genes including ORF17, ORF0, and ORF23 are absent in the OsAdV genome and in those of other aviadenoviruses of non-domestic birds. The genome organization and gene order of OsAdV is similar to those of OlAdV, but with some differences. The ORF14 and ORF19 genes of both genomes have paralogs (ORF14A and ORF19A). OsAdV has one fiber gene encoding a protein of 818 aa, which is the largest fiber gene of all aviadenoviruses and the second largest one found in an adenovirus, after the fiber of deer mastadenovirus B, which is 1010 aa long. OlAdV has two fiber genes, which encode proteins of 405 and 745 aa, respectively. Alignment of the amino acid sequences of the OsAdV and OlAdV fiber proteins showed that the long fiber of OlAdV has potential homology to the fiber of OsAdV. The first 350 aa of the two proteins have a similar sequence (52% aa sequence identity). The region from aa 351 to 818 of OsAdV is highly divergent and fails to make a successful alignment with aa 351-745 of OlAdV using the NCBI BLASTp method. Like most reported adenoviruses, OsAdV has 22-kDa and 33-kDa proteins. By contrast, OlAdV has a 33-kDa protein but lacks a 22-kDa protein. A small ORF with coding capacity for a 54-aalong protein was found near the left end of the genome. A BLASTx search with this ORF failed to find any matches in the NR and SWISS-PROT databases. We provisionally designate this ORF as a putative gene for a hypothetical protein.
In summary, we have determined the complete genome sequence of a putative new aviadenovirus from owls. A closely related virus belonging to the same lineage was Each virus is labelled with its NCBI accession number, serotype, and species found in Japan and sequenced previously. An interesting feature of the Japanese isolate was that chicks of more than six different owl species that were infected with this virus were found dead at a breeding facility. Because adult birds that were kept outdoors had no clinical signs, researchers hypothesized that vertical transmission might have occurred. Our novel virus was obtained from cloacal samples from free-living, apparently healthy scops owls. Further studies on the ability of this virus to infect owls belonging to other species and its possible pathogenicity to young birds would be of interest.