Comparative Hemagglutinin Phylogeny and Whole-Genome Molecular Characterization of Avian Influenza Viruses Circulating in Romania with Markers of Pathogenicity and Host Adaptation

Avian influenza A(H5) viruses of clade 2.3.4.4b continue to circulate in European wild and domestic bird populations, posing sustained threats to avian and public health. Romania occupies an epidemiologically important position along major migratory flyways, representing a focal point for repeated virus introductions. This study characterized the genetic diversity and molecular markers of H5 avian influenza viruses detected in Romania during 2024–2026 and placed them within a broader European evolutionary context. We screened complete eight-segment genomes from 31 Romanian H5 viruses collected between 3 January 2024 and 24 March 2026 for molecular markers and analyzed their hemagglutinin genes using maximum-likelihood phylogenetics alongside 618 contemporary hemagglutinin sequences from Hungary, Bulgaria, Serbia, Ukraine, Italy, and Germany. Phylogenetic analysis of the hemagglutinin gene placed Romanian viruses within European clade 2.3.4.4b diversity, forming strongly supported clusters with viruses from neighboring countries. All 31 viruses possessed the multibasic hemagglutinin cleavage-site motif PLREKRRKR/GLFG, consistent with the highly pathogenic molecular profile. The PB2 E627K substitution was identified in one virus (3.2%); no other mammalian-adaptation marker examined was detected in this virus, and D701N, T271A and Q591R/K were absent from the entire dataset. Molecular screening of all eight genomic segments revealed a predominantly conserved amino acid background with limited variation at selected positions. These results contribute to the molecular epidemiology of highly pathogenic avian influenza H5 in south-eastern Europe, and support continued whole-genome surveillance.