Isolation and Whole Genome Sequencing of an Avian Orthoavulavirus 16 from Cinereous Vulture (Aegypius monachus) in South Korea, 2023

Yongwoo Son¹Sun-Hak Lee^1,2,3*Dong-Yeop Lee¹Hyunjun Cho⁴Yemoon Son⁴Daehun Kim⁴Chang-seon Song³Dong-Hun Lee¹

• ¹Wildlife Health Laboratory, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea
• ²Konkuk University, Seoul, Republic of Korea
• ³Avian disease laboratory, College of Veterinary Medicine, Konkuk University, Seoul, Republic of Korea
• ⁴National Institute of Wildlife of Disease Control and Prevention, Gwangju, Republic of Korea

Avian avulaviruses (AAVVs) encompass a diverse group of negative-sense, single-stranded RNA viruses within the subfamily Avulavirinae (family Paramyxoviridae), currently comprising 22 recognized serotypes (APMV-1 to APMV-22) that predominantly circulate in wild and domestic avian species worldwide (1,2). While Avian orthoavulavirus 1 (AOAV-1)-the causative agent of Newcastle disease-remains the most clinically and economically significant due to its potential for high virulence in poultry (3), the majority of non-AOAV-1 serotypes, including Avian orthoavulavirus 16 (AOAV-16), are generally considered low-pathogenic or asymptomatic in their natural hosts (4)(5)(6).Orthoavulavirus upoense, known as AOAV-16, was first characterized in 2014 from fecal samples of overwintering wild birds in South Korea, marking its initial detection in East Asia (6). Subsequent genomic surveillance efforts identified AOAV-16 in wild waterfowl across Central Asia, including a virus archived in 2006 from Kazakhstan, suggesting a broader distribution along migratory flyways connecting East Asia, Siberia, and other regions across Eurasia (7,8). AOAV-16 contains a nonsegmented ~15.1-kb negative-sense RNA genome arranged in the canonical avulavirus gene order (3′-nucleoprotein (NP)-phosphoprotein (P)-matrix protein (M)-fusion protein (F)-hemagglutininneuraminidase (HN)-large polymerase protein (L)-5′) (6). Phylogenetic analyses based on complete genome sequences and NP gene sequences showed AOAV-16 is genetically closest to class I lentogenic lineages commonly isolated from Anseriformes, supporting a possible shared evolutionary origin and ecological association (7,8). Despite these findings from surveillance efforts, genomic resources for AOAV-16 remain extremely scarce, with only a handful of complete or near-complete genomes available in public databases. This scarcity constrains robust inference regarding viral evolution, host range, recombination potential, and risk of spillover into domestic poultry. Moreover, most confirmed AOAV-16 detections have originated from wild waterfowl, underscoring uncertainties surrounding its host range breadth and the potential involvement of non-Anseriform species in viral maintenance and dissemination.Here, we report the first coding-complete genome sequence of AOAV-16 from a fecal dropping of cinereous vulture (Aegypius monachus), a scavenging raptor, found) collected in South Korea in 2023. This detection expands highlights the presence of AOAV-16 genomic RNAthe recognized host range of AOAV-16 beyond aquatic birds, and into including Accipitriformes, a taxonomic group not previously associated with this serotype. The genome sequences and their phylogenetic relationships serve as valuableprovide reference data for future APMV surveillance and research. A fecal sample was collected from a fallow paddy field near Geum River, South Korea (GPS coordinates ≈ 36.0455°N, 126.7434°E) on December 26, 2023, by National Institute of Wildlife Disease Control and Prevention (NIWDC) as part of the national wild bird surveillance program for avian influenza virus in South Korea. The sample was inoculated into 10-day-old embryonated chicken eggs. Allantoic fluid was tested by hemagglutination assay and screened for influenza A virus by real-time quantitative reverse transcription polymerase chain reactions (qRT-PCR) using Qiagen Quantitect RT-PCR reagents (Qiagen, Manchester, UK), following a previously described protocol (9). Hemagglutination-positive but influenza-negative allantoic fluid was filtered through a 0.22 µm syringe filter, and RNA was extracted from allantoic fluid using the RNeasy Mini Kit (Qiagen, Germany). The RNA was subjected to semi-nested RT-PCR targeting the paramyxovirus large protein (L) gene (10). The host species was identified by mitochondrial cytochrome oxidase I (COI) DNA barcoding of the fecal sample (11). For whole-genome sequencing, viral RNA was amplified by sequence-independent single-primer amplification (SISPA) (12), and a sequencing library was prepared using Illumina DNA Prep kit. Sequencing was performed on a MiniSeq platform (Illumina, USA). Metagenomic classification was performed using CZID (13) to identify viral reads and guide reference sequence selection. Following adapter and SISPA-primer removal with BBDuk ( 14) and Geneious Prime 2025.2.2 (https://www.geneious.com), quality-filtered reads were assembled with using Minimap2 with Orthoavulavirus upoense (GenBank accession no. OR270139) as reference and deposited to GenBank (accession no. PX482736). Complete genome sequences of avulavirus references designated by International Committee on Taxonomy of Viruses (ICTV) were retrieved from GenBank. Maximum likelihood (ML) phylogenetic trees were reconstructed in IQ-TREE v3.0.1 under the best-fit substitution model determined by ModelFinder (15). Branch support was assessed with 1,000 replicates of Shimodaira-Hasegawa approximate likelihood ratio test and ultrafast bootstrap. Trees were visualized using iTOL v7 (16). Each CDS of AOAV-16 was pairwise compared at the nucleotide level to calculate sequence identity.To examine genetic diversity within AOAV-16, all available complete genome and F gene sequences were retrieved from GenBank and analyzed using the same ML approach. Temporal phylogenetic analysis of F gene was performed in BEAST v10.5.0 under the TN93+F+I model, selected by ModelFinder, with an uncorrelated lognormal relaxed clock and a Gaussian Markov Random Field (GMRF) Bayesian skyride coalescent prior (17). Five independent Markov chain Monte Carlo (MCMC) runs of 50 million generations each were combined after removal of 10% burn-in. Convergence was confirmed using Tracer v1.7.2 (http://tree.bio.ed.ac.uk/software/tracer/), and the maximum clade credibility (MCC) tree was generated with TreeAnnotator and visualized in FigTree v1.4.5 (http://tree.bio.ed.ac.uk/software/figtree/). This study reports the eleventh coding-complete genome of AOAV-16 deposited in public databases (GenBank accession no. PX482736) and the first from a non-Anseriformes host. Using highthroughput next-generation sequencing (NGS), we generated a 15,139-nucleotide coding-complete genome for AOAV-16/cinereous_vulture/South_Korea/23WF447-15P/2023 (hereafter, vulturederived AOAV-16) with a mean sequencing depth of 17,151×. Length The length of the complete CDS was 14,904-nt, consistent with the paramyxovirus "rule of six" (18). Metagenomic classification using CZID identified reads mapping to avulaviruses, thereby supporting AOAV-16 as the predominant viral species in the sample and guiding selection of the reference genome for assembly.The genome exhibited the canonical avulavirus gene order (3'-NP-P-M-F-HN-L-5'), and the F protein cleavage site was monobasic (¹¹⁰LVQAR↓L¹¹⁵), a motif conserved across all known AOAV-16 isolates and indicative of low virulence (Fig. 1B). Notably, the HN-to-L intergenic region contained a 12-nt deletion in the hexanucleotide repeat array (AAAAAU)ₙ compared to other AOAV-16 strains, as confirmed by raw read mapping (8). Mitochondrial DNA barcoding confirmed the host species of the fecal sample as cinereous vulture.Phylogenetic analysis confirmed that the vulture-derived isolate clustered monophyletically within the AOAV-16 clade, which is most closely related to AOAV-1 at the species level (Fig. 1A).Pairwise ) with high posterior probability support (Fig. 2). However, the broad 95% Bayesian credible intervals surrounding the divergence time estimates (tMRCA: September 1989, 95% HPD: 4 September 1961 to 30 September 2004) are likely attributable to the limited number of available genome sequences. Consequently, these estimates should be interpreted cautiously. However, The the tMRCA of the root was inferred to be 1989-9-22, but thewith a wide 95% Bayesian credible interval was wide,( 1961-9-4 to 2004-9-30), due to thereflecting limited amount of sequence data availability. The mean substitution rate was 7.66 × 10⁻⁴ substitutions/site/year (s/s/y, 95% HPD: 1.84 × 10⁻⁴ -1.52 × 10⁻³).⁻³) which was similar to values reported for other lentogenic avulaviruses, including AOAV-1 (1.78 × 10⁻³ s/s/y) and AOAV-4 (1.29 × 10⁻³ s/s/y) (19,20). Nucleotide divergence among available AOAV-16 genomes ranged from 0.14% to 3.2% despite a decade-long temporal and geographic span. We assume that this pattern mirrors that of other lentogenic avulaviruses such as AOAV-8 ( 21), a pattern that may be consistent with limited selective pressure in wild hostssuggesting limited selective pressure in asymptomatically infected wild hosts. The geographic clustering of all AOAV-16 viruses within the East Asian-Australasian Flyway (EAAF) suggests sustained transmission along this migratory corridor. However, virus propagation in embryonated chicken eggs prior to sequencing may have introduced selection bias, potentially favoring egg-adapted variants over the original fecal viral population (19).The isolation of AOAV-16 from a fecal droppings of a cinereous vulture-a scavenging raptor in the order Accipitriformes-represents the first detection in a non-Anseriformes host. This finding challenges the prevailing view of AOAV-16 as waterfowl-restricted and raises questions about transmission dynamics. Potential routes include direct exposure to infected waterfowl at shared wetlands, consumption of contaminated carcasess, or transient mucosal infection during scavenging. As apex predators and scavengers, raptors may serve as sentinel species or ecological bridges, facilitating gene flow between aquatic and terrestrial avian communities -a role increasingly recognized in the epidemiology of other avian viruses, including influenza A (23).Taken together, the present findings warrant cautious interpretation but suggest that inclusion of non-traditional avian species-particularly scavenging species such as cinereous vultures-may help capture a broader range of viral detection contexts. Further longitudinal and multi-host studies at shared habitats will be required to clarify their relevance to transmission dynamics and poultry spillover risk.In conclusion, this first report of AOAV-16 in an Accipitriformes host, supported by whole-genome sequencing, significantly advances our understanding of viral ecology and host plasticity. This study underscores the value of integrating non-traditional hosts into surveillance strategies, particularly in biodiversity hotspots like South Korea. While monitoring has predominantly targeted waterfowl along migratory flyways, including raptors-especially scavengers like cinereous vultures-may improve detection of cryptically circulating AOAVs. Future efforts should prioritize longitudinal sampling at shared habitats to elucidate transmission networks and assess poultry spillover risk. All authors declare that this research was conducted without any commercial or financial relationships that could be interpreted as potential conflicts of interest.