As a unique antigenic variant of Newcastle disease virus (NDV) in pigeons, genotype VI NDVs cause serious disease in pigeons and pose a potential threat to domestic poultry. In this study, analyses of genetic variation and evolution of global genotype VI NDVs revealed the worldwide distribution and continuous evolution of genotype VI NDVs. Of importance, VI.2.1.1.2.1 and VI.2.1.1.2.2 are the two prevalent sub-genotypes worldwide. The virulence, replication capacity, and pathogenesis in chickens of representative strains from VI.2.1.1.2.1 and different clusters in VI.2.1.1.2.2 were further evaluated. Compared with the VI.2.1.1.2.1 strain, increased replication capacity and virulence in chickens were observed in strains of VI.2.1.1.2.2. VI.2.1.1.2.2 strains showed preferential binding for α-2,3-linked sialic acids and superior performance in viral entry, cell-cell fusion, and release of progeny virions. Evaluation with recombinant viruses demonstrated that residues at positions 365 and 497 in the HN protein contributed to the differences in biological characteristics of VI.2.1.1.2.2 strains, and residue 365 was a key determinant. Moreover, our results showed that the activity of the viral replication complex contributed to the differences in replication capacity among these viruses, with the P protein being the major individual contributor. The optimal effect was achieved when the NP and L proteins were homologous. Moreover, the 5' terminal trailer region was also found to be involved in the replication capacity of genotype VI NDVs; however, the viral V protein was not related to the replication and virulence of these viruses. Our findings highlight the potential risk of VI.2.1.1.2.2 NDVs due to their persistent circulation and evolution.

Importance: Genotype VI is the most diverse group of Newcastle disease viruses (NDVs). In addition to infectious disease in pigeons, the potential threat to chicken flocks and the public health implications associated with genotype VI NDVs also need to be addressed. Herein, comprehensive genetic evolution analysis revealed a global distribution pattern and continuous evolution of genotype VI NDVs worldwide. The biological characteristics of genotype VI NDVs belonging to different sub-genotypes were also evaluated. In particular, the widespread transmission, circulation, and constant evolution of currently prevalent sub-genotype VI.2.1.1.2.2 have led to the alteration of receptor binding preference, an increase in replication capacity, and a resultant increase in virulence in chickens. These findings expand our current understanding of the evolution and pathogenesis of genotype VI NDVs.

Keywords: Newcastle disease virus; genetic evolution; replication; sub-genotype VI.2.1.1.2.2; virulence.