Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1

Abstract

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.

Keywords: Adenoviridae; Atadenovirus; Passerine adenovirus-1; evolution; genome organisation.

Figure 1

Schematic illustration of the avian atadenoviruses. Schematic map of the passerine adenovirus 1 (PaAdV-1, GenBank accession no. MT674683), in comparison with duck adenovirus A (DAdV-A, GenBank accession no. AC_000004) and psittacine adenovirus 3 (PsAdV-3, GenBank accession no. KJ675568), using CLC Genomic Workbench (version 9.5.4, CLC bio, a QIAGEN Company, Prismet, Aarhus C, Denmark). The arrows symbolize adenovirus genes and open reading frames (ORFs) predicted to code for proteins, indicating their direction of transcription. Each gene or ORF is colour coded, as indicated by the colour key in the legend. The bottom graph represents the sequence conservation between the aligned PaAdV-1, DAdV-A, and PsAdV-3 sequences at a given coordinate at each position in the alignment. The gradient of the colour reflects the conservation of that particular position is in the alignment. Red presents 100% conservation across all three viruses, black 50% conserved regions, and blue less than 50% conserved regions.

Figure 2

Predicted structure of the unique PaAdV1-ORF10. (A) prediction of transmembrane helices (TMHs) in unique PaAdV1-ORF10 gene using EMBOSS 6.5.7 tool in Geneious (version 10.2.2) (A), TMHMM (B), and TMpred (C). All the programs consistently predicted two TMHs. (A) TMHs detected by EMBOSS also showed the presence of alpha-helices within TMHs predicted region that has been dominated by highly hydrophobic residue (red colour). (B,C) The x-axis represents the position of residue, whereas y-axis represents the posterior probability (B), and scores (above 500 are considered significant) (C) for the predicted TMHs. (C) Solid and dashed black lines indicate protein orientation as inside to outside, and outside to inside, respectively.

Figure 3

Predicted structure of the unique PaAdV1-ORF18. (A) prediction of transmembrane helices (TMHs) in unique PaAdV1-ORF18 gene, using EMBOSS 6.5.7 tool in Geneious (version 10.2.2) (A), TMHMM (B), and TMpred (C). All the programs consistently predicted two TMHs. (A) TMHs detected by EMBOSS also showed the presence of alpha-helices within TMHs predicted region that has been dominated by highly hydrophobic residue (red colour). (B,C) The x-axis represents the position of residue, whereas the y-axis represents the posterior probability (B) and scores (above 500 were considered significant) (C) for the predicted TMHs. (C) Solid and dashed black lines indicate protein orientation as inside to outside, and outside to inside, respectively.

Figure 4

Phylogenetic tree shows the possible evolutionary relationship of novel passerine adenovirus 1 with other selected AdVs. Maximum likelihood (ML) tree was constructed by using concatenated amino acid sequences of the complete DNA-dependent DNA polymerase, pTP, penton, and hexon genes. Concatenated protein sequences were aligned with MAFTT (version 7.450) [41] in Geneious (version 10.2.2, Biomatters, Ltd., Auckland, New Zealand), under the BLOSUM62 scoring matrix and gap open penalty = 1.53. The gap >20 residues deleted from the alignments. The unrooted ML tree was constructed with PhyML [42] under the LG substitution model, and 1000 bootstrap re-samplings were chosen to generate ML trees, using tools available in Geneious (version 10.2.2, Biomatters, Ltd., Auckland, New Zealand). The numbers on the left show bootstrap values as percentages, and the labels at branch tips refer to original AdVs species name, followed by GenBank accession number in parentheses. The final tree is visualised with FigTree (version 1.4.4) [43]. The five official genera are highlighted as different background colours, and novel passerine adenovirus 1 is shown in pink colour.