Evidence of a Possible Viral Host Switch Event in an Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi)

Avipoxviruses have been characterized from many avian species. Two recent studies have reported avipoxvirus-like viruses with varying pathogenicity in reptiles. Avipoxviruses are considered to be restricted to avian hosts. However, reports of avipoxvirus-like viruses from reptiles such as the green sea turtle (Chelonia mydas) and crocodile tegu (Crocodilurus amazonicus) suggest that cross-species transmission, within avian species and beyond, may be possible. Here we report evidence for a possible host switching event with a fowlpox-like virus recovered from an endangered northern royal albatross (Diomodea sanfordi)—a species of Procellariiformes, unrelated to Galliformes, not previously known to have been infected with fowlpox-like viruses. Complete genome sequencing of this virus, tentatively designated albatrosspox virus 2 (ALPV2), contained many fowlpox virus-like genes, but also 63 unique genes that are not reported in any other poxvirus. The ALPV2 genome contained 296 predicted genes homologous to different avipoxviruses, 260 of which were homologous to an American strain of fowlpox virus (FWPV). Subsequent phylogenetic analyses indicate that ALPV2 likely originated from a fowlpox virus-like progenitor. These findings highlight the importance of host-switching events where viruses cross species barriers with the risk of disease in close and distantly related host populations.


Introduction
Over the past several decades, marine bird populations have been decreasing globally [1] with the sustainability of the albatrosses (family Diomedeidae) and large petrels (Macronectes and Procellaria spp.) being under significant threat [2][3][4]. One of the world's largest seabirds, the northern royal albatross (Diomedea sanfordi) is categorized as an "endangered" species under the International Union for Conservation of Nature (IUCN) Red List and is classified as Category B for management urgency [5]. Northern royal albatrosses range extensively throughout the Southern Ocean, yet infrequently into Antarctic waters. Breeding colonies are confined to the Chatham Islands and Taiaroa Head on the Otago Peninsula, Dunedin, New Zealand. The overall breeding population in the Chatham Islands colonies (99% of the total) is anticipated at about 6500-7000 pairs, which equates to a total population of approximately 17,000 adults [6].
Human activities, including commercial fishing and pollution, are recognized as threats for incidental mortality of these species [5,[7][8][9][10][11]. Invasive alien species, degradation or destruction of nesting grounds, storms and flooding, pollution of the marine environment, and ingestion of plastics are additional factors contributing to population

Genome Assembly and Annotation
The resulting 3,374,936 paired-end raw sequence reads were used to assemble the complete genome of ALPV2, using CLC Genomics Workbench (version 9.5.4, CLC bio, a QIAGEN Company, Prismet, Aarhus C, Denmark) and Geneious (version 10.2.2, Biomatters, New Zealand), as described previously [29][30][31][32][33]. This resulted in the generation of a 286,155 bp genome. Clean raw reads (2.38 million) were mapped back to the assembled ALPV2 genome resulting in an average coverage of 668.55x. The genome was annotated according to the previously published protocol [18] using Geneious software (version 10 Biomatters, New Zealand). Open reading frames (ORFs) longer than 30 amino acids, with a methionine start codon (ATG) and minimal overlap with other ORFs (not exceeding 50% of one of the genes), were selected and annotated. ORFs shorter than 30 amino acids that had been previously annotated in other poxvirus genomes were also included. Similarity BLAST searches were performed on the predicted ORFs and were annotated as potential genes if predicted ORFs showed significant sequence similarity to known viral or cellular genes (BLAST E value ≤ e −5 ) [34].
To predict the function of putative unique ORFs identified in this study, the derived protein sequence of each ORF was searched using multiple applications to identify conserved domains or motifs. Transmembrane helices were searched using the TMHMM package (version 2.0) [35] and TMpred [36]. Additionally, searches for conserved secondary structure (HHpred) [37] and protein homologs were conducted using Phyre2 [38] and SWISS-MODEL [39].

Comparative Genomics
Genomic features of the newly sequenced ALPV2 were visualised using Geneious (version 10.2.2). Sequence similarity percentages between representative Chordopoxvirus (ChPV) and ALPV2 complete genome sequences were determined using tools available in Geneious (version 10.2.2). Dot plots were created based on the EMBOSS dottup program in Geneious software, with word size = 12 [40].

Phylogenetic Analyses
Phylogenetic analyses were performed using the ALPV2 genome sequence determined in this study, together with other selected ChPV genome sequences available in GenBank (Table 1). Nucleotide sequences of the partial DNA polymerase and partial p4b genes, as well as concatenated amino acid sequences of nine poxvirus core proteins, were aligned as described previously [41] using the MAFTT L-INS-I algorithm implemented in Geneious (version 7.388) [42]. To determine the best-fit model to construct phylogenetic analyses, a model test was performed using CLC Genomics Workbench (version 9.5.4), which favoured a general-time-reversible model with gamma distribution rate variation and a proportion of invariable sites (GTR+G+I). Phylogenetic analyses for nucleotide sequences were performed under the GTR substitution model, but the WAG substitution model was chosen for concatenated amino acid sequences with 1000 bootstrap replicates in CLC Genomic Workbench (version 9.5.4).
Comparison of the ALPV2 genome with those of other avipoxviruses was performed using dot plot analyses. The ALPV2 genome was highly syntenic with FWPV, PEPV, FGPV and FeP2 (Figure 2A-D). Moreover, differences in synteny with FGPV and FeP2 were observed ( Figure 2C-D, highlighted as black arrows), mainly due to the presence of two large additional copies of variola B22R gene family proteins in ALPV2 (ORF-170 and -171, 1870 and 1766 amino acids in length, respectively). The ALPV2 genome also demonstrated significant differences in the entire genome compared to ALPV, SWPV1, Comparison of the ALPV2 genome with those of other avipoxviruses was performed using dot plot analyses. The ALPV2 genome was highly syntenic with FWPV, PEPV, FGPV and FeP2 (Figure 2A-D). Moreover, differences in synteny with FGPV and FeP2 were observed ( Figure 2C-D, highlighted as black arrows), mainly due to the presence of two large additional copies of variola B22R gene family proteins in ALPV2 (ORF-170 and -171, 1870 and 1766 amino acids in length, respectively). The ALPV2 genome also demonstrated significant differences in the entire genome compared to ALPV, SWPV1, CNPV and TKPV ( Figure 2E-H). Within these highlighted regions (orange arrows), multiple SNPs and insertions/deletions (indels) accounted for the variation observed between the genomes ( Figure 2E-H).
CNPV and TKPV ( Figure 2E-H). Within these highlighted regions (orange arrows), multiple SNPs and insertions/deletions (indels) accounted for the variation observed between the genomes ( Figure 2E-H).  Table 1 for virus details and GenBank accession numbers). The Classic color scheme was chosen in Geneious (version 10.2.2) for the dot plot lines according to the length of the match, from blue for short matches to red for matches over 100 bp long. Window size = 12.

Core/conserved ORFs
Similarly to other ChPVs, the ALPV2 genome contained 87 conserved core genes, which are involved in essential functions such as replication, transcription and virion assembly (Supplementary Table S1; highlighted with bold and italic font). The number of conserved ChPV genes is considered to range between 83-90 [24,51,57,58], which is consistent with the findings in the ALPV2 genome. Among them, two of the predicted ORFs, encoding immunodominant virion proteins ALPV-231 and -232, were truncated compared to FWPV168, which may warrant further studies to determine whether they are expressed and functional. Based on a recent study by Carulei et al. [24], we also searched for a further 47 genes that are conserved in avipoxviruses ( Table 3). The ALPV2 genome was also predicted to contain these 47 conserved ORFs (Table 3), and none of the genes were found to be truncated or fragmented compared to a closely related fowlpox virus (FWPV) [45].  Table 1 for virus details and GenBank accession numbers). The Classic color scheme was chosen in Geneious (version 10.2.2) for the dot plot lines according to the length of the match, from blue for short matches to red for matches over 100 bp long. Window size = 12.

Core/Conserved ORFs
Similarly to other ChPVs, the ALPV2 genome contained 87 conserved core genes, which are involved in essential functions such as replication, transcription and virion assembly (Supplementary Table S1; highlighted with bold and italic font). The number of conserved ChPV genes is considered to range between 83-90 [24,51,57,58], which is consistent with the findings in the ALPV2 genome. Among them, two of the predicted ORFs, encoding immunodominant virion proteins ALPV-231 and -232, were truncated compared to FWPV168, which may warrant further studies to determine whether they are expressed and functional. Based on a recent study by Carulei et al. [24], we also searched for a further 47 genes that are conserved in avipoxviruses ( Table 3). The ALPV2 genome was also predicted to contain these 47 conserved ORFs (Table 3), and none of the genes were found to be truncated or fragmented compared to a closely related fowlpox virus (FWPV) [45].

Multigene Families
Avipoxviruses are the largest ChPVs and contain several, large, multigene families with immune related functions comprising up to 50% of the genome [24,51]. Table 4 shows the copy numbers of each of the 14 multigene families identified in the ALPV2 genome compared with the other selected sequenced avian poxvirus genomes, including the recently characterized genomes of ALPV, MPPV2 and PEPV2. ALPV2 has a similar complement of multigene families compared to FWPV (total of 95 and 89 gene copies, respectively). However, ALPV2 has a significantly lower number of multigene families in comparison with ALPV (GenBank accession no. MW365933.1) (total of 95 and 139 gene copies, respectively). The copy number of ankyrin repeat, N1R/p28, C-type lectin and TGF-β family genes were significantly higher in the ALPV2 genome compared to ALPV.

Evolutionary Relationships of ALPV2
Phylogenetic reconstruction using concatenated amino acid sequences of selected conserved ChPV genes provides clear evidence for the inclusion of ALPV2 in the genus Avipoxvirus. In the maximum likelihood (ML) tree (Figure 3), ALPV2 was located within a sub-clade A1 encompassing mostly FWPV isolates from chickens and wild turkeys with 100% bootstrap support. Using the same set of concatenated protein sequences, we found that the maximum inter-lineage sequence identity values ranged from 99% to 100% among FWPV isolates from chickens and turkeys, and ALPV2, respectively, which illustrated the phylogenetic position of this avipoxvirus sequenced from an endangered northern royal albatross, and further inferred that these viruses were likely derived from a common progenitor. Strikingly, a few other avipoxviruses were also positioned in the same A1 subclade when we used partial nucleotide sequences of the DNA polymerase and p4b genes ( Figure 4). This included poxviruses isolated from birds of the order Galliformes (domestic fowl, Gallus domesticus; blue-eared pheasant, Crossoptilon auritum) in Hungary and Hawaii, respectively [59], and a Psittaciformes (superb parrot, Polytelis swainsonii) originating from Chile [59], which are almost identical to ALPV2 within this relatively small fragment of the genome.   Table 1. Saltwater crocodile poxvirus 1 (SwCRV1; MG450915) [53] was used as an outgroup. Major clades and sub-clades are designated according to Gyuranecz et al. (2013) [59].

Discussion
We have documented a second avipoxvirus infection in a northern royal albatross, notably involving a possible host switch event. Applying various approaches for genomic comparison, ALPV2 was shown to be genetically similar to other avipoxviruses with the highest nucleotide sequence similarity being to an American virulent strain of fowlpox virus (99.3%), followed by PEPV (76.6%), FGPV (75.0%), and FeP2 (73.1%). The AT content of the ALPV2 genome was 69.1%, which was consistent with previously reported avipoxviruses. Given the phylogenetic relationship of ALPV2 (Figures 3 and 4; Supplementary Figures S1 and S2), we propose that this virus, in contrast to ALPV [29], originated from a common ancestor that deviated from a fowlpox virus-like progenitor. Notably, there were significant differences observed between the ALPV2 and ALPV genomes (regarding nucleotide identity, genome length and number of ORFs (Table 2)), confirming that two distinct avipoxviruses were present in juvenile northern royal albatrosses sampled on the Otago Peninsula (South Island of New Zealand) in March 1997.
Interestingly, the phylogenetic relationship of ALPV2 using concatenated protein sequences encoded by the chosen poxvirus core genes (Figure 3), and partial nucleotide sequences of DNA polymerase and P4b genes (Figure 4), demonstrate that the newly assembled albatrosspox virus genome is representative of a sub-clade A1 avipoxvirus. In support of our findings, nearly identical partial nucleotide sequences of fowlpox viruses were isolated from other non-chicken avian species including blue-eared pheasant (Crossoptilon auritum) in Hawaii [59] and a Psittaciformes (superb parrot, Polytelis swainsonii) originating from Chile [59], which are also positioned in the same A1 sub-clade. In addition, an atypical fowlpox virus was also reported from a fatally ill rhinoceros in 1969 [28]; however, genetic characterization of this virus has not been reported. Two recent studies of avipoxvirus-like viruses provide examples of viruses able to infect non-avian hosts: cheloniid poxvirus 1 (ChePV-1) and teiidaepox virus 1 (TePV-1), which have been linked with infections in green sea turtle and crocodile tegu [26,27]. Our findings suggest an unusual transmission event may have occurred, resulting in lesions, representing either a "dead-end" event and/or that fowlpox-like viruses may have a wider host range with the ability to cross chicken-host barriers. Characterization of additional avipoxviruses isolated from these endangered albatross species may enable the likelihood and importance of cross species transmission of fowlpox and fowlpox-like viruses to be determined.
It is not possible to explain the host-pathogen dynamics of ALPV2 from this case alone, but it is apparent that northern royal albatrosses and other avian species share an ecological habitat. Northern royal albatrosses are typically solitary foragers, however they may gather at food sources at sea. Most of their food is thought to be obtained by feeding on dead or dying prey from the surface and by scavanging fish, rubbish and bait discarded from commercial fishing boats. This may indicate a possible scenario for virus transmission that requires further investigation. Another potential route of virus transmission is mechanical, where biting arthropods are assumed to play a part in the spread of avipoxviruses within wild bird populations. Ticks, fleas [60], hippoboscid flies [61], and mosquitos [19,62] may all play a role as mechanical vectors. Northern royal albatrosses also congregate at breeding colonies where transmission of avipoxviruses by insect vectors is likely to occur.
Avipoxviruses have been reported to infect other albatross species, including a critically endangered Waved Albatross (Phoebastria irrorata) [63] and Laysan Albatross (Phoebastria immutabilis) [21]. The severity of avipoxvirus infections is variable and infrequently fatal, although secondary bacterial and fungal infections may subsequently occur and cause mortality [23]. Nevertheless, the pathogenic effects of avipoxvirus infections in seabirds are not adequately understood, and the longer-term consequences in susceptible populations remain to be determined. The genetic information provided in this study will facilitate improved understanding of the evolution of poxviruses in this avian species, thus contributing to the development of improved management and conservation strategies for the endangered northern royal albatross.

Conclusions
We have reported the genomic characterization of a second novel avipoxvirus, tentatively designated ALPV2, isolated from an endangered northern royal albatross on the Otago Peninsula, New Zealand, and provide evidence that this infection resulted from a possible host switch event. The ALPV2 genome was similar to those of other avipoxviruses and likely originated from a common ancestor that deviated from a fowlpox virus-like progenitor. This finding has increased our knowledge of the pathogen diversity within northern royal albatrosses in New Zealand. However, additional investigations will be required to better understand relevant host-pathogen dynamics including routes of transmission and factors leading to infection, associated pathology and disease prevalence.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/v14020302/s1, Figure S1: Maximum likelihood (ML) phylogenetic tree from partial nucleotide sequences of the DNA polymerase gene of selected avipoxviruses; Figure  S2: Maximum likelihood (ML) phylogenetic tree from partial nucleotide sequences of the P4b gene of selected avipoxviruses; Table S1: Albatrosspox virus 2 (ALPV2) genome annotations and comparative analysis of ORFs.

Institutional Review Board Statement:
The material used in this study was provided by New Zealand Ministry for Primary Industries, Animal Health Laboratory, which was submitted for diagnostic purposes. The findings from the diagnostic material were to be used in a publication, and no specific ethical approval was required.

Informed Consent Statement: Not applicable.
Data Availability Statement: The complete genome sequence and associated datasets generated during this study were deposited in GenBank under the accession number OK348853.