Quasispecies Diversity and Amino Acid Variation at the Fusion Protein Cleavage Site of Newcastle Disease Virus Isolated from Cormorants in Central Asia

Newcastle disease virus (NDV) remains a major threat to poultry production worldwide, and wild waterbirds are recognized as reservoirs of virulent strains. To characterize fine-scale quasispecies diversity at the virulence-determining fusion (F) protein cleavage site, amplicons encompassing the six-amino-acid motif were deep-sequenced from seven egg-passaged cormorant-derived isolates. Fifty-four distinct amino-acid substitutions passed predefined abundance and statistical filters and were detected across all six cleavage-site positions. The cleavage-site quasispecies was dominated by a limited set of recurrent substitutions (R1G, F6L, F6V, F6C, R4G, and several position-2 variants), each supported by hundreds of thousands of reads and occurring at frequencies of 1–26% across samples. Pooled Shannon entropy indicated that positions 1 and 6 were the most variable, whereas positions 3 and 5 showed lower diversity across the dataset. Principal component analysis showed apparent differences in mutation-frequency profiles among the seven egg-passaged isolates; the first two principal components explained 97.9% of the variance (PC1, 87.4%; PC2, 10.5%). Heatmap visualization showed that a subset of variants, including R1G and F6L/V/C, was consistently abundant across samples, whereas others were isolate-specific. These data describe position-specific cleavage-site heterogeneity in seven egg-passaged NDV isolates originally recovered from cormorants. The observed variant frequencies may reflect a combination of diversity present in the original specimens and changes introduced or selected during egg amplification. The coexistence of the predominant velogenic RRQRRF motif with lower-frequency alternative and stop-codon-containing haplotypes documents sequence heterogeneity in outbreak-related material after egg amplification and provides a descriptive basis for future genotype-resolved and phenotype-linked studies.