Newcastle disease virus (NDV) can infect over 250 bird species with variable pathogenicity; it can also infect humans in rare cases. The present study investigated an outbreak in feral pigeons in São Paulo city, Brazil, in 2019. Affected birds displayed neurological signs, and hemorrhages were observed in different tissues. Histopathology changes with infiltration of mononuclear inflammatory cells were also found in the brain, kidney, proventriculus, heart, and spleen. NDV staining was detected by immunohistochemistry. Twenty-seven out of thirty-four tested samples (swabs and tissues) were positive for Newcastle disease virus by RT-qPCR test, targeting the M gene. One isolate, obtained from a pool of positive swab samples, was characterized by the intracerebral pathogenicity index (ICPI) and the hemagglutination inhibition (HI) tests. This isolate had an ICPI of 0.99, confirming a virulent NDV strain. The monoclonal antibody 617/161, which recognizes a distinct epitope in pigeon NDV strains, inhibited the isolate with an HI titer of 512. A complete genome of NDV was obtained using next-generation sequencing. Phylogenetic analysis based on the complete CDS F gene grouped the detected isolate with other viruses from subgenotype VI.2.1.2, class II, including one previously reported in Southern Brazil in 2014. This study reports a comprehensive characterization of the subgenotype VI.2.1.2, which seems to have been circulating in Brazilian urban areas since 2014. Due to the zoonotic risk of NDV, virus surveillance in feral pigeons should also be systematically performed in urban areas.

Pigeon paramyxovirus 1 (PPMV-1) comprises pigeon-associated variants of Newcastle disease virus (NDV), predominantly belonging to genotypes VI, XX, and XXI. These viruses have been detected in feral pigeons in Brazil since at least 2014, and an NDV outbreak associated with a pigeon-origin genotype VI virus was reported in commercial broilers in 2024. Here, we characterized two PPMV-1 strains detected in pigeons samples from geographically distinct regions of Brazil. Virus BR/105/21 was detected in Midwestern Brazil, whereas BR/34/22 was detected in Southeastern Brazil. Genome sequencing yielded a complete genome sequence for BR/105/21 (15,192 nt; 100% reference-genome coverage; mean depth, 3334×) and a near-complete genome sequence for BR/34/22 (15,133 nt; 99.5% reference-genome coverage; mean depth, 20.8×). Both viruses possessed the F-protein cleavage-site motif ^113RQKR↓F^117, which is consistent with the molecular criterion for virulent NDV. Phylogenetic analysis based on the complete F-gene coding sequence grouped both viruses with PPMV-1 previously detected in Brazil between 2014 and 2019. Analysis of VI.2.1.2 sequences identified three well-supported phylogenetic groups with between-group nucleotide distances of 6.1–8.0%. Based on established NDV classification criteria, two groups are proposed as VI.2.1.2.1, comprising viruses from Nigeria detected between 2007 and 2015, and VI.2.1.2.2, comprising Brazilian viruses detected between 2014 and 2022 and one virus detected in Argentina in 1997. These findings expand the available genetic data for pigeon-associated NDV in South America and reveal substantial genetic heterogeneity and phylogenetic structure within VI.2.1.2, reinforcing the importance of genomic surveillance of NDV in pigeons.