Avian paramyxoviruses (APMVs) belong to the subfamily Avulavirinae, family Paramyxoviridae, and currently define 22 different avulavirus species (APMV-1 to APMV-22) that are further divided into three genera. Metaavulavirus (APMV-2, -5 to -8, -10, -11, -14, -15, -20, and -22), Orthoavulavirus (APMV-1, -9, -12, -13, -16 to -19, and -21), and Paraavulavirus (APMV-3 and -4) according to the International Committee on Taxonomy of Viruses (ICTV) (Young et al., 2022). APMVs are negative-sense, single-stranded RNA viruses (Lee et al., 2017). Most APMVs encode at least six proteins, namely nucleoprotein (N), phosphoprotein (P), matrix (M) protein, fusion (F) protein, hemagglutinin-neuraminidase (HN) protein, and large polymerase (L), which are arranged in the order of 3’ leader, N, P, M, F, HN, L and 5’ trailer (Samal, 2011). Among these, the F protein is involved with the entry of the virus, and the HN protein has receptor-binding and neuraminidase activity and contributes to viral replication (Kim et al., 2011; Morrison, 2003). Partial or complete nucleotide sequences of the F gene have been commonly employed in phylogenetic analyses for genotype classification (Diel et al., 2012).

Specific APMV serotypes are important in the poultry industry due to their ability to cause clinical disease in domestic birds (Saif, 2009). APMV-1, known as Newcastle disease virus (NDV), is globally distributed, and its highly pathogenic forms cause substantial economic losses to the poultry industry (Alexander et al., 2012). APMV-1 is classified based on clinical severity and molecular characterization of the amino acid sequences at the F protein cleavage site. Velogenic strains, characterized by high virulence, possess a multi-basic cleavage site motif, whereas lentogenic strains exhibit low virulence and contain a monobasic motif (Seal et al., 2005; Klink et al., 2023). While the remaining APMV serotypes are generally regarded as low or asymptomatic virulence in poultry, APMV-2, APMV-3, APMV-4, APMV-6, and APMV-7 have been reported to cause mild clinical signs in domestic birds (Nayak et al., 2012; Yin et al., 2017; Alexander, 2000).

Wild birds are known as natural reservoirs of both APMVs and influenza A viruses (Thomas et al., 2008). During seasonal migration, wild birds play a pivotal role in the intercontinental dissemination of viruses across geographic regions (Treshchalina et al., 2022). To date, nine major migratory bird flyways have been recognized worldwide, serving as critical routes for seasonal wild birds’ movements (Pearce-Higgins et al., 2017). South Korea is located in the East Asian-Australasian Flyway (EAAF), acting as a key migratory corridor and breeding site for numerous migratory bird species (Son et al., 2024). In particular, the western regions of South Korea serve as major wintering grounds for migratory birds, with increased wild bird abundance during autumn migration and winter (Lee et al., 2020). These western regions are characterized by high densities of both wild birds and poultry, creating favorable conditions for the circulation of avian influenza (AI) viruses (Song et al., 2017). In response, active AI virus surveillance programs targeting migratory birds have been conducted in South Korea since 2012, contributing to a better understanding of APMV distribution and diversity among wild birds in South Korea (Jeong et al., 2018). Several APMV serotypes, including APMV-1, APMV-4, APMV-6, APMV-13, APMV-14, APMV-15, and APMV-21, have been detected in wild waterfowl migrating through South Korea (Lee et al., 2024; Choi et al., 2013; Lee et al., 2017; Choi et al., 2018; Kuhn et al., 2020; Youk et al., 2025). Notably, lentogenic APMV-1 strains isolated from domestic ducks in South Korea exhibited high genetic similarity to virulent NDV strains previously identified in wild waterfowl in China, implying possible interregional transmission (Gaikwad et al., 2016). Furthermore, APMV-4 has been isolated not only from wild birds but also from domestic ducks, supporting the possibility of interspecies transmission between wild and domestic avian populations (Tseren-Ochir et al., 2017). These findings underscore the importance of continued surveillance of APMVs in wild birds to understand their role in APMV epidemiology better and to inform effective prevention strategies.

Recent advances in high-throughput sequencing technologies have significantly facilitated investigations into the genetic diversity and evolutionary dynamics of APMVs (Dimitrov et al., 2019). Accordingly, this study employed a next-generation sequencing (NGS) approach using single-primer amplification (SISPA) to obtain complete genome sequences and assess the genetic characteristics of APMVs from wild bird fecal samples collected in South Korea between 2020 and 2024.

This study expands our understanding of the genetic diversity and molecular characteristics of APMVs circulating in migratory wild birds in South Korea between 2020 and 2024. A total of 27 APMV isolates were obtained from wild birds during the winter seasons between 2020 and 2024, with a mean isolation rate of 0.35%. This result is comparable to a previous study from South Korea, which reported a mean APMV isolation rate of 0.4% during the winter seasons from 2015 to 2021 (Lee et al., 2024). All isolates were derived from Anseriformes species, which aligns with previous studies and demonstrates that these birds serve as a natural reservoir of APMVs (Gulyaeva et al., 2022; Cross et al., 2013). These findings highlight the importance of continued surveillance efforts targeting Anseriformes species to improve understanding of the ecology and evolution of APMVs.

Four distinct APMV serotypes were identified, and coding-complete genome sequences were obtained using a SISPA approach. Recent advances in high-throughput sequencing technologies have markedly improved the availability of complete genome data for previously underrepresented APMV serotypes (Van Borm et al., 2023). SISPA-based next-generation sequencing has been effectively applied in previous studies to generate complete APMV genomes and was similarly employed in this investigation (Chrzastek et al., 2017; Kariithi et al., 2022; Cho et al., 2022). However, the 5’ and 3’ UTRs were only partially recovered in most isolates. This limitation is consistent with a previous study reporting that SISPA-based approaches often fail to resolve the terminal 100-200 nucleotides of viral UTRs (Tal et al., 2019). In this study, the limited sequencing depth (approximately 100,000 reads per sample) may have contributed to the insufficient coverage required for complete UTR recovery. To enhance the recovery of full-length UTRs, the use of NGS platforms with higher read output or the incorporation of rapid amplification of cDNA ends (RACE)-PCR might be beneficial.

APMV-1 continues to receive particular attention due to its established pathogenic potential and significant implications for the poultry industry. Newcastle disease, caused by virulent strains of APMV-1, was first reported in South Korea in 1927 and has remained a significant concern for the national poultry industry (Kim and Song, 1992). Although no outbreaks have been recorded in domestic poultry farms in South Korea in recent years, APMV-1 is still regularly detected in wild bird populations (Choi et al., 2012; Lee et al., 2024). In the present study, 12 APMV-1 isolates were obtained and classified into class I sub-genotype 1.2 and class II sub-genotype I.2. Based on the classification system proposed by Diel et al. (2012), the class I sub-genotype 1.2 viruses were further divided into sub-genotype 1c and 1d (Diel et al., 2012). While sub-genotype 1d has predominantly been reported in North America, sub-genotype 1c includes strains originating from diverse regions, including Europe, East Asia, and Central Asia (Hicks et al., 2019). However, our findings suggest that sub-genotype 1d is not limited to North America. In particular, 23JBN-F22-66-70, classified as sub-genotype 1d, showed high nucleotide similarity to East Asian strains, indicating that this lineage also includes viruses of East Asian origin. This observation suggests the ongoing intercontinental circulation driven by migratory bird movements. Although Class II sub-genotype I.2 strains are typically lentogenic and detected in wild waterfowl, possible transmission to domestic poultry has been suggested in previous studies (Lee et al., 2024; Reid et al., 2023). In this study, no evidence of Class II sub-genotype I.2 virus transmission from wild waterfowl to domestic poultry was identified, although careful monitoring remains essential.

All 12 APMV-1 isolates identified in this study possessed a monobasic cleavage site in the F protein. Representative class I sub-genotype 1.2 isolates, including 23JBN-F22-66-70 and 22JBN-F2-10, exhibited MDT of > 168 h, which is consistent with the presence of a monobasic cleavage site. In contrast, 21JBN-F27-27 exhibited MDT of 89.6 h and was classified as mesogenic despite also possessing a monobasic cleavage site. This finding contrasts with the general understanding that mesogenic NDVs have a polybasic cleavage site motif within the F protein (Xiao et al., 2012). These findings suggest that MDT-based pathogenicity does not always strictly correlate with the F protein cleavage site motif. Although MDT alone is insufficient for assessing viral pathogenicity and additional animal experiments would be required, the higher MDT-based pathogenicity observed for 21JBN-F27-27 relative to other APMV-1 isolates suggests that class II sub-genotype 1.2 viruses warrant further investigation in surveillance and pathogenicity studies.

Lentogenic live vaccines based primarily on genotypes I and II are currently widely employed worldwide for Newcastle disease control. The F and HN amino acid sequences of the isolated APMV-1 strains were compared with those of representative vaccine strains, including V4 (genotype I) and LaSota (genotype II), both of which are commonly used as lentogenic live vaccines (Hu et al., 2022). Considering the crucial role of F and HN proteins in eliciting protective immune responses, identifying amino acid differences in key functional domains may provide insight into potential antigenic variation and implications for vaccine efficacy (Kumar et al., 2011). The results showed that while class II sub-genotype I.2 isolates exhibited conserved amino acid sequences, class I sub-genotype 1.2 isolates exhibited notable amino acid diversity, especially within the F protein. Amino acid substitutions in the F gene have been suggested to influence antigenic properties, which may have implications for current vaccine performance (Shahar et al., 2018). In support of this, antigenic cross-reactivity assays using anti-NDV LaSota serum demonstrated uniformly low HI titers (≤ 20) against class I sub-genotype 1.2 isolates. Notably, low HI titers were also observed for class II sub-genotype I.2 isolates despite their relatively conserved F and HN amino acid sequences, indicating limited antigenic cross-reactivity with the NDV vaccine strain. Although these findings suggest antigenic divergence between wild bird-derived APMV-1 isolates and commonly used NDV vaccine strain, further studies, including virus neutralization assays and in vivo vaccination—challenge experiments, are required to determine whether currently commercialized vaccines provide effective protection against APMV-1 isolates originating from wild birds.

According to a recent phylogenetic classification based on MCC tree analyses of the F gene, APMV-4 is grouped into four major genotypes (I–IV), with Genotype I further subclassified into three distinct sub-genotypes (Zhang et al., 2021). Previously identified APMV-4 isolates in South Korea were predominantly classified into Genotype I (formerly referred to as the Old-World I genotype), which includes both Eurasian and Asian sub-genotypes, as well as Genotype IV (formerly referred to as the Old-World II genotype). In this study, APMV-4 strains belonging to both the Eurasian sub-genotype of Genotype I and to Genotype II were identified. The APMV-4 isolates exhibited high nucleotide similarity to strains previously reported in China, Russia, and Taiwan. Notably, five APMV-4 isolates belonged to Genotype II, a lineage referred to as the New-World I genotype, which includes APMVs primarily isolated from wild birds in the Americas. To date, no Genotype II APMV-4 strains have been reported from wild birds in South Korea. The identification of Genotype II APMV-4 strains in this study suggests potential interhemispheric exchange between the Eastern and Western Hemispheres, with novel APMV-4 genotypes that might be introduced into South Korea through migratory flyways.

The cleavage sites of all APMV-4 F genes lacked multiple basic residues, indicating lentogenic viruses. In addition, consistent with a previous study, the hypervariable regions of the F protein in APMV-4 isolates from this study were relatively conserved (Zhang et al., 2021). However, APMV-4 has been documented not only in wild birds but also in domestic poultry such as ducks, geese, and chickens (Zhang et al., 2021; Yin et al., 2017; Tseren-Ochir et al., 2017). In addition, given the potential for APMV-4 transmission from wild birds to domestic poultry, continued surveillance is warranted in South Korea, even when APMV-4 is detected only in wild bird populations (Yin et al., 2017).

APMV-6 has been classified into two genotypes, G1 and G2, based on phylogenetic analysis (Choi et al., 2018; Young et al., 2022). Interestingly, G2 shows substantial genetic divergence from G1 and has even been proposed as a putative novel species cluster (Young et al., 2022). In South Korea, the two genotypes have also demonstrated distinctive ecological characteristics (Choi et al., 2018). In the present study, one APMV-6 strain (22JBN-F4-34) belonging to the G1 genotype was isolated and characterized by a SH protein that distinguishes it from other APMV serotypes. 22JBN-F4-34 showed high nucleotide similarity to isolates from wild birds in neighboring countries such as China, Taiwan, and Russia, suggesting possible cross-regional circulation of APMV-6.

Nevertheless, due to the limited availability of genetic data, a comprehensive phylogenetic relationship between the genotypes G1 and G2 remains unclear. Sequence analysis of the F gene cleavage site in 22JBN-F4-34 indicated a lentogenic APMV. Nonetheless, experimental evidence from an APMV-6 strain isolated in South Korea has demonstrated its ability to infect poultry and shed virus, highlighting the importance of sustained monitoring efforts (Lee et al., 2013).

APMV-13 is known to circulate at low levels among wild bird populations and has been reported less frequently compared to other APMV serotypes (Lee et al., 2024). The 22JBN-F14-46-50 exhibited the highest nucleotide similarity to a previously reported strain, Greater white-fronted goose/South Korea/E20-158-3/2020. Consistent with other APMV-13 strains, 22JBN-F14-46-50 possessed a lentogenic-type fusion protein cleavage site motif (VRENRL). Notably, the 22JBN-F14-46-50 also exhibited an 84-nucleotide deletion at positions 15,304–15,387, which was identical to that observed in the E20-158-3 strain (Cho et al., 2022). Furthermore, an identical deletion was also identified in another Korean isolate, E21-954 (accession number: PV643987), detected in a wild bird in Korea in 2021. These results suggest the possibility of emerging genomic diversity within APMV-13 strains currently circulating in South Korea. However, due to the limited availability of full-length genomic sequences of APMV-13, further studies are needed to clarify the evolution and genetic diversity of APMV-13.

APMVs have been continuously reported in migratory wild birds in South Korea, including a variety of subtypes and even novel serotypes (Lee et al., 2024; Lee et al., 2017). In the present study, we provide the first molecular evidence of a genotype II APMV-4 strain in wild birds from South Korea, thereby expanding the current understanding of the genetic diversity and evolutionary complexity of APMVs circulating in East Asia. In addition, class I sub-genotype 1.2 of APMV-1 isolates identified in this study exhibited multiple amino acid substitutions in the F protein when compared with commonly used NDV vaccine strains, which may have implications for vaccine efficacy. These results highlight the importance of ongoing surveillance of APMV-1 strains circulating in wild bird populations. Moreover, regardless of serotype, phylogenetic studies provide evidence of active intercontinental exchanges of APMVs. The nearly complete nucleotide sequences obtained from diverse APMV subtypes provide valuable information that will contribute to future investigations into the genetic evolution, antigenic variability, and epidemiological dynamics of APMVs.