The P protein T25M substitution is involved in the quasispecies and virulence of Newcastle disease virus

ABSTRACT The non-virulent Newcastle disease virus (NDV)-Spotted Dove strain acquires enhanced virulence after serial passage in chicken embryos. In this study, the P gene T25M substitution was identified as the critical determinant of increased pathogenicity after 100 consecutive passages in specific-pathogen-free (SPF) chicken embryos. Initial characterization of the parental Spotted Dove strain confirmed a lentogenic phenotype by mean death time (MDT) in 9-day-old SPF embryos, intracerebral pathogenicity index (ICPI) in 1-day-old chicks, intravenous pathogenicity index (IVPI) in 6-week-old chickens, limited replication in DF-1 cells, and minimal tissue pathology. The virus exhibited shortened MDT, elevated ICPI and IVPI scores, higher replication in DF-1 and BHK-21 cells, and extensive lesions in respiratory and neural tissues. Next-generation sequencing of five viral RNA samples from the parental strain and selected passage points revealed six nonsynonymous mutations, with only T25M in P correlating with the virulence phenotype. To validate its role, recombinant NDV-Dove strains (rDove) and rDove 100th -P T25M were generated and co-inoculated into 9-day-old SPF chicken embryos at 1 × 10 4 PFU/mL. Similarly, rDove and rDove 100th -P T25M containing green and red fluorescence, respectively, were co-infected into DF-1 cells at a multiplicity of infection of 0.01 with equal ratios (1:1, 1:9, and 9:1). After 10 passages, results indicated that multiple factors equalized the quasispecies frequencies as the initial differences between “C” and “T” were gradually overcome by the evolutionary processes of the viral population in chicken embryos and DF-1 cells. Our findings identify P-T25M as the key adaptive mutation driving enhanced NDV virulence and illustrate the complex quasispecies evolution during serial passage. IMPORTANCE This research investigated the T25M mutation in the P gene of the Newcastle disease virus (NDV)-Spotted Dove strain, which emerged as a significant mutation conferring virulence after 100 serial passages through chicken embryos. The P gene typically encodes a viral phosphoprotein that serves various roles in the viral life cycle, including involvement in viral RNA synthesis, interaction with other viral proteins, and occasionally modulation of the host immune response. A T25M mutation, characterized by the substitution of threonine with methionine at position 25, has the potential to modify the structural configuration of the P gene. The findings highlight the significance of the extensive quasispecies population of NDV, which develops virulence and improves viral replication and transmission in chickens. Key point mutations, particularly T25M in these cases, are essential for viral fitness and the evolution of NDV quasispecies in chicken embryos and DF-1 cells.