A Review on Highly Pathogenic Avian Influenza H5N1 Clade 2.3.4.4b in Mammals: Molecular Adaptation, Host Range Expansion, and One Health Implications

Abstract

Influenza A virus H5N1 clade 2.3.4.4b has caused the largest highly pathogenic avian influenza (HPAI) panzootic on record, infecting over 175 million poultry globally and spilling over into more than 60 mammalian species. The establishment of reassortant genotype B3.13 in US dairy cattle from March 2024, with 1,093 confirmed herds across 19 states by April 2026, is the first sustained HPAI transmission in a domestic ruminant and defines the pandemic risk landscape of the current era. Two mechanistically independent adaptive axes drive bovine fitness in B3.13. The polymerase axis, encompassing PB2 M631L and PA K497R, remodels the viral replication complex to exploit host-specific ANP32A isoforms. The receptor axis, driven by hemagglutinin (HA) mutations D104G and V147M, enables efficient recognition of N-glycolylneuraminic acid, which comprises approximately half the sialic acid pool in bovine mammary tissue but is absent in humans. Crystallographic analysis has established that a single Q226L substitution in the HA receptor-binding site of a human clinical isolate of bovine H5N1 is sufficient to switch its receptor specificity from avian-type to human-type receptors, identifying this position as the highest-priority genomic surveillance target. Three pandemic barriers remain intact as of early 2026: B3.13 retains avian-type receptor preference, an elevated HA fusion pH, and full nucleoprotein (NP) sensitivity to human MxA restriction. Even so, partial ferret respiratory-droplet transmission, over 70 confirmed human cases, including the first US pediatric case, the emergence of a baloxavir-resistant clinical isolate, and ongoing convergent molecular evolution collectively define a pandemic risk level higher than any prior point in H5N1 history. Integrated One Health action combining mandatory bulk-milk genomics, wastewater surveillance, systematic wildlife serosurveillance, and accelerated ruminant mRNA vaccination is urgently required.