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Newcastle disease virus (NDV) infects a wide range of hosts, including poultry and wild birds. However, most research has focused on poultry-derived strains, with limited studies on the biological characteristics of NDV isolated from wild birds. This study investigated genotype IX NDV strains from wild birds. Genetic evolution and viral ecology analyses suggest possible poultry-to-wild-bird transmission of this genotype. Most of the genotype IX NDV strains, isolated from asymptomatic wild birds, were highly pathogenic in chickens, but one strain isolated from spotted dove exhibited attenuated virulence. This dove-derived strain showed the highest homology with a virulent strain from Eurasian blackbird, differing by only 12 amino acids across proteins. Using reverse genetics, we identified the viral HN protein as a critical determinant of virulence in the dove strain. The coexistence of F110 and G116 residues in the HN protein was associated with reduced HN cell-surface abundance and downstream decreases in viral membrane fusion activity, replication capacity, and tissue tropism. Computational modeling further suggests that the F110/G116 combination may be linked to a local α-helix within the HN head-stalk linker region, providing a plausible structural context for these effects. Crucially, this dove strain's fusion protein retained the furin-cleavable, virulent-type "¹¹²RRQRRF¹¹⁷" cleavage site. Immunizing chickens with this strain induced high antibody titers within 1 week, and titers persisted at high levels for 13 weeks and provided complete protection against virulent challenge at both early and late post-immunization time points. Our findings uncover an HN-linked attenuation mechanism centered on residues 110 and 116, primarily through their effects on HN cell-surface abundance and downstream fusion activity, offering new insights into the development of immunogenic NDV vaccines with reduced virulence.IMPORTANCEWild birds, as natural reservoirs of Newcastle disease virus (NDV), harbor a diverse range of viral strains. However, research on these strains remains limited. Here, we show that genotype IX NDV detected in wild birds most likely reflects possible poultry-to-wild bird transmission rather than long-term endemic circulation in wild populations. Using isolates derived from wild birds, we report a novel attenuation mechanism involving structural modification of the HN protein's head-neck linker region. Therefore, research on wild bird-derived isolates not only provides insights into the epidemiological dynamics of NDV transmission but also facilitates the identification of novel attenuated strains, which could aid in the development of high-efficacy NDV vaccines and NDV-based viral vectors.